Hepatitis C virus inhibitors

ABSTRACT

This disclosure concerns novel compounds of Formula (I) or Formula (II) as defined in the specification and compositions comprising such novel compounds. These compounds are useful antiviral agents, especially in inhibiting the function of the NS5A protein encoded by Hepatitis C virus (HCV). Thus, the disclosure also concerns a method of treating HCV related diseases or conditions by use of these novel compounds or a composition comprising such novel compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 61/168,891 filed Apr. 13, 2009.

FIELD OF THE DISCLOSURE

The present disclosure is generally directed to antiviral compounds, and more specifically directed to compounds which can inhibit the function of the NS5A protein encoded by Hepatitis C virus (HCV), compositions comprising such compounds, and methods for inhibiting the function of the NS5A protein.

BACKGROUND OF THE DISCLOSURE

HCV is a major human pathogen, infecting an estimated 170 million persons worldwide—roughly five times the number infected by human immunodeficiency virus type 1. A substantial fraction of these HCV infected individuals develop serious progressive liver disease, including cirrhosis and hepatocellular carcinoma.

The current standard of care for HCV, which employs a combination of pegylated-interferon and rivbavirin, has a non-optimal success rate in achieving sustained viral response and causes numerous side effects. Thus, there is a clear and long-felt need to develop effective therapies to address this undermet medical need.

HCV is a positive-stranded RNA virus. Based on a comparison of the deduced amino acid sequence and the extensive similarity in the 5′ untranslated region, HCV has been classified as a separate genus in the Flaviviridae family. All members of the Flaviviridae family have enveloped virions that contain a positive stranded RNA genome encoding all known virus-specific proteins via translation of a single, uninterrupted, open reading frame.

Considerable heterogeneity is found within the nucleotide and encoded amino acid sequence throughout the HCV genome due to the high error rate of the encoded RNA dependent RNA polymerase which lacks a proof-reading capability. At least six major genotypes have been characterized, and more than 50 subtypes have been described with distribution worldwide. The clinical significance of the genetic heterogeneity of HCV has demonstrated a propensity for mutations to arise during monotherapy treatment, thus additional treatment options for use are desired. The possible modulator effect of genotypes on pathogenesis and therapy remains elusive.

The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins. In the case of HCV, the generation of mature non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one is believed to be a metalloprotease and cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (also referred to herein as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions by both acting as a cofactor for the NS3 protease and assisting in the membrane localization of NS3 and other viral replicase components. The formation of a NS3-NS4A complex is necessary for proper protease activity resulting in increased proteolytic efficiency of the cleavage events. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B (also referred to herein as HCV polymerase) is a RNA-dependent RNA polymerase that is involved in the replication of HCV with other HCV proteins, including NS5A, in a replicase complex.

Compounds useful for treating HCV-infected patients are desired which selectively inhibit HCV viral replication. In particular, compounds which are effective to inhibit the function of the NS5A protein are desired. The HCV NS5A protein is described, for example, in the following references: S. L. Tan, et al., Virology, 264:1-12 (2001); K.-J. Park, et al., J. Biol. Chem., 30711-30718 (2003); T. L. Tellinghuisen, et al., Nature, 435, 374 (2005); R. A. Love, et al., J. Virol, 83, 4395 (2009); N. Appel, et al., J. Biol. Chem., 281, 9833 (2006); L. Huang, J. Biol. Chem., 280, 36417 (2005); C. Rice, et al., WO2006093867.

SUMMARY OF THE DISCLOSURE

The present disclosure provides compounds which selectively inhibit HCV viral replication, as characterized by Formula (I) or Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is

R¹ is hydrogen or —C(O)R^(x);

R² is hydrogen or —C(O)R^(y);

R^(x) and R^(y) are each independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more, preferably one to three, substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(c)R^(d),

wherein any said aryl and heteroaryl may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, OR⁵, —NR^(a)R^(b), (NR^(a)R^(b)) alkyl, and (MeO)(HO)P(O)O—, and

wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴;

R³ is hydrogen, alkyl, or arylalkyl;

R⁴ is alkyl or arylalkyl;

R⁵ is hydrogen, alkyl, or arylalkyl;

R^(a) and R^(b) are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, —C(O)R⁶, —C(O)OR⁷, —C(O)NR^(c)R^(d), and (NR^(c)R^(d))alkyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, aryl, hydroxyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, and halogen;

R⁶ is alkyl;

R⁷ is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and

R^(c) and R^(d) are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.

The compounds of the present disclosure can be effective to inhibit the function of the HCV NS5A protein. In particular, the compounds of the present disclosure can be effective to inhibit the HCV 1b genotype or multiple genotypes of HCV. Therefore, this disclosure also encompasses: (1) compositions comprising a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and (2) a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF THE DISCLOSURE

In a first aspect of the present disclosure compounds of Formula (I) or Formula (II) are provided:

In a first embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is

R¹ is hydrogen or —C(O)R^(x);

R² is hydrogen or —C(O)R^(y);

R^(x) and R^(y) are each independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more, preferably one to three, substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(c)R^(d),

wherein any said aryl and heteroaryl may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, OR⁵, —NR^(a)R^(b), (NR^(a)R^(b)) alkyl, and (MeO)(HO)P(O)O—, and

wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴;

R³ is hydrogen, alkyl, or arylalkyl;

R⁴ is alkyl or arylalkyl;

R⁵ is hydrogen, alkyl, or arylalkyl;

R^(a) and R^(b) are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, —C(O)R⁶, —C(O)OR⁷, —C(O)NR^(c)R^(d), and (NR^(c)R^(d))alkyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, aryl, hydroxyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, and halogen;

R⁶ is alkyl;

R⁷ is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and

R^(c) and R^(d) are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.

In a second embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), wherein Z is oxygen, further characterized by Formula (Ia) or Formula (IIa):

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is

L² is

R¹ is hydrogen or —C(O)R^(x);

R² is hydrogen or —C(O)R^(y);

R^(x) and R^(y) are each independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more, preferably one to three, substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(c)R^(d),

wherein any said aryl and heteroaryl may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, OR⁵, —NR^(a)R^(b), (NR^(a)R^(b))alkyl, and (MeO)(HO)P(O)O—, and

wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴;

R³ is hydrogen, alkyl, or arylalkyl;

R⁴ is alkyl or arylalkyl;

R⁵ is hydrogen, alkyl, or arylalkyl;

R^(a) and R^(b) are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, —C(O)R⁶, —C(O)OR⁷, —C(O)NR^(c)R^(d), and (NR^(c)R^(d))alkyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, aryl, hydroxyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, and halogen;

R⁶ is alkyl;

R⁷ is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and

R^(c) and R^(d) are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.

In a third embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), wherein Z is sulfur, further characterized by Formula (Ib) or Formula (IIb):

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is

L² is

R¹ is hydrogen or —C(O)R^(x);

R² is hydrogen or —C(O)R^(y);

R^(x) and R^(y) are each independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more, preferably one to three, substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(c)R^(d),

wherein any said aryl and heteroaryl may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, OR⁵, —NR^(a)R^(b), (NR^(a)R^(b))alkyl, and (MeO)(HO)P(O)O—, and

wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴;

R³ is hydrogen, alkyl, or arylalkyl;

R⁴ is alkyl or arylalkyl;

R⁵ is hydrogen, alkyl, or arylalkyl;

R^(a) and R^(b) are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, —C(O)R⁶, —C(O)OR⁷, —C(O)NR^(c)R^(d), and (NR^(c)R^(d))alkyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, aryl, hydroxyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, and halogen;

R⁶ is alkyl;

R⁷ is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and

R^(c) and R^(d) are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.

In a fourth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), wherein Z is NH, further characterized by Formula (Ic) or Formula (IIc):

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is

L² is

R¹ is hydrogen or —C(O)R^(x);

R² is hydrogen or —C(O)R^(y);

R^(x) and R^(y) are each independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more, preferably one to three, substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(c)R^(d),

wherein any said aryl and heteroaryl may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, OR⁵, —NR^(a)R^(b), (NR^(a)R^(b))alkyl, and (MeO)(HO)P(O)O—, and

wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴;

R³ is hydrogen, alkyl, or arylalkyl;

R⁴ is alkyl or arylalkyl;

R⁵ is hydrogen, alkyl, or arylalkyl;

R^(a) and R^(b) are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, —C(O)R⁶, —C(O)OR⁷, —C(O)NR^(c)R^(d), and (NR^(c)R^(d))alkyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, aryl, hydroxyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, and halogen;

R⁶ is alkyl;

R⁷ is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and

R^(c) and R^(d) are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.

In a fifth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), wherein L¹ is

and L² is

further characterized by Formula (Id) or Formula (IId):

or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

R¹ is hydrogen or —C(O)R^(x);

R² is hydrogen or —C(O)R^(y);

R^(x) and R^(y) are each independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more, preferably one to three, substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(a)R^(d),

wherein any said aryl and heteroaryl may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, OR⁵, —NR^(a)R^(b), (NR^(a)R^(b)) alkyl, and (MeO)(HO)P(O)O—, and

wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴;

R³ is hydrogen, alkyl, or arylalkyl;

R⁴ is alkyl or arylalkyl;

R⁵ is hydrogen, alkyl, or arylalkyl;

R^(a) and R^(b) are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, —C(O)R⁶, —C(O)OR⁷, —C(O)NR^(c)R^(d), and (NR^(c)R^(d))alkyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, aryl, hydroxyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, and halogen;

R⁶ is alkyl;

R⁷ is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and

R^(c) and R^(d) are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.

In a sixth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), wherein L¹ is

and L² is

further characterized by Formula (Ie) or Formula (IIe):

or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

R¹ is hydrogen or —C(O)R^(x);

R² is hydrogen or —C(O)R^(y);

R^(x) and R^(y) are each independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more, preferably one to three, substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(c)R^(d),

wherein any said aryl and heteroaryl may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, OR⁵, —NR^(a)R^(b), (NR^(a)R^(b))alkyl, and (MeO)(HO)P(O)O—, and

wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴;

R³ is hydrogen, alkyl, or arylalkyl;

R⁴ is alkyl or arylalkyl;

R⁵ is hydrogen, alkyl, or arylalkyl;

R^(a) and R^(b) are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, —C(O)R⁶, —C(O)OR⁷, —C(O)NR^(c)R^(d), and (NR^(c)R^(d))alkyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, aryl, hydroxyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, and halogen;

R⁶ is alkyl;

R⁷ is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and

R^(c) and R^(d) are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.

In a seventh embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), wherein L¹ is

and L² is

further characterized by Formula (Ig) or Formula (IIg):

or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

R¹ is hydrogen or —C(O)R^(x);

R² is hydrogen or —C(O)R^(y);

R^(x) and R^(y) are each independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more, preferably one to three, substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(c)R^(d),

wherein any said aryl and heteroaryl may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, OR⁵, —NR^(a)R^(b), (NR^(a)R^(b))alkyl, and (MeO)(HO)P(O)O—, and

wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴;

R³ is hydrogen, alkyl, or arylalkyl;

R⁴ is alkyl or arylalkyl;

R⁵ is hydrogen, alkyl, or arylalkyl;

R^(a) and R^(b) are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, —C(O)R⁶, —C(O)OR⁷, —C(O)NR^(c)R^(d), and (NR^(c)R^(d))alkyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, aryl, hydroxyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, and halogen;

R⁶ is alkyl;

R⁷ is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and

R^(c) and R^(d) are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.

In an eighth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), wherein L¹ is

and L² is

further characterized by Formula (Ih) or Formula (IIh):

or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

R¹ is hydrogen or —C(O)R^(x);

R² is hydrogen or —C(O)R^(y);

R^(x) and R^(y) are each independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more, preferably one to three, substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(c)R^(d),

wherein any said aryl and heteroaryl may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, OR⁵, —NR^(a)R^(b), (NR^(a)R^(b)) alkyl, and (MeO)(HO)P(O)O—, and

wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴;

R³ is hydrogen, alkyl, or arylalkyl;

R⁴ is alkyl or arylalkyl;

R⁵ is hydrogen, alkyl, or arylalkyl;

R^(a) and R^(b) are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, —C(O)R⁶, —C(O)OR⁷, —C(O)NR^(c)R^(d), and (NR^(c)R^(d))alkyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, aryl, hydroxyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, and halogen;

R⁶ is alkyl;

R⁷ is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and

R^(c) and R^(d) are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.

In a ninth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is

R¹ is —C(O)R^(x);

R² is —C(O)R^(y);

R^(x) and R^(y) are independently alkyl substituted by at least one —NR^(a)R^(b), characterized by Formula (A):

wherein:

m is 0 or 1;

R⁸ is hydrogen or alkyl;

R⁹ is selected from hydrogen, cycloalkyl, aryl, heteroaryl, heterocyclyl, and alkyl optionally substituted with a substituent selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, heterobicyclyl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(c)R^(d),

wherein any said aryl and heteroaryl may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, haloalkyl, arylakyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, —OR⁵, —NR^(a)R^(b), (NR^(a)R^(b)) alkyl, and (MeO)(HO)P(O)O—, and

wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more, preferably one to three, substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴;

R³ is hydrogen, alkyl, or arylalkyl;

R⁴ is alkyl or arylalkyl;

R⁵ is hydrogen, alkyl, or arylalkyl;

R^(a) and R^(b) are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, —C(O)R⁶, —C(O)OR⁷, —C(O)NR^(c)R^(d), and (NR^(c)R^(d))alkyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, aryl, hydroxyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, and halogen;

R⁶ is alkyl;

R⁷ is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and

R^(c) and R^(d) are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.

In a tenth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is

R¹ is —C(O)R^(x);

R² is —C(O)R^(y);

R^(x) and R^(y) are independently alkyl substituted by at least one —NR^(a)R^(b), characterized by Formula (A):

wherein:

m is 0;

R⁸ is hydrogen or C₁ to C₄ alkyl;

R⁹ is selected from hydrogen, C₁ to C₆ alkyl optionally substituted with —OR¹², C₃ to C₆ cycloalkyl, allyl, —CH₂C(O)NR^(c)R^(d), (NR^(c)R^(d)) alkyl,

wherein j is 0 or 1;

k is 1, 2, or 3;

n is 0 or an integer selected from 1 through 4;

each R¹⁰ is independently hydrogen, C₁ to C₄ alkyl, C₁ to C₄ haloalkyl, halogen, nitro, —OBn, or (MeO)(OH)P(O)O—;

R¹¹ is hydrogen, C₁ to C₄ alkyl, or benzyl;

R¹² is hydrogen, C₁ to C₄ alkyl, or benzyl;

R^(a) is hydrogen or C₁ to C₄ alkyl;

R^(b) is C₁ to C₄ alkyl, C₃ to C₆ cycloalkyl, benzyl, 3-pyridyl, pyrimidin-5-yl, acetyl, —C(O)OR⁷, or —C(O)NR^(c)R^(d);

R⁷ is C₁ to C₄ alkyl or C₁ to C₄ haloalkyl;

R^(c) is hydrogen or C₁ to C₄ alkyl; and

R^(d) is hydrogen, C₁ to C₄ alkyl, or C₃ to C₆ cycloalkyl.

In an eleventh embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is

R¹ is —C(O)R^(x);

R² is —C(O)R^(y);

R^(x) and R^(y) are independently alkyl substituted by at least one —NR^(a)R^(b), characterized by Formula (A):

wherein:

m is 0;

R⁸ is hydrogen;

R⁹ is phenyl optionally substituted with one up to five substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, halogen, C₁ to C₆ alkoxy, hydroxyl, cyano, and nitro; and

NR^(a)R^(b) is a heterocyclyl or heterobicyclyl group selected from:

wherein n is 0, 1, or 2;

each R¹³ is independently selected from C₁ to C₆ alkyl, phenyl, trifluoromethyl, halogen, hydroxyl, methoxy, and oxo; and

R¹⁴ is C₁ to C₆ alkyl, phenyl, benzyl, or —C(O)OR¹⁵ group, wherein R¹⁵ is C₁ to C₄ alkyl, phenyl, or benzyl.

In a twelfth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is

R¹ is —C(O)R^(x);

R² is —C(O)R^(y);

R^(x) and R^(y) are independently alkyl substituted by at least one —NR^(a)R^(b), characterized by Formula (A):

wherein:

m is 1;

R⁸ is hydrogen;

R⁹ is C₁ to C₆ alkyl, arylalkyl, or heteroarylalkyl;

R^(a) is hydrogen; and

R^(b) is —C(O)OR⁷, wherein R⁷ is C₁ to C₆ alkyl.

In a thirteenth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is

R¹ is —C(O)R^(x);

R² is —C(O)R^(y); and

R^(x) and R^(y) are heteroaryl or heterocyclyl independently selected from:

wherein n is 0 or an integer selected from 1 through 4;

each R¹³ is independently selected from hydrogen, C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, phenyl, benzyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, heterocyclyl, halogen, NR^(c)R^(d), hydroxyl, cyano, and oxo, where R^(c) and R^(d) are independently hydrogen or C₁ to C₄ alkyl; and

R¹⁴ is hydrogen (H), C₁ to C₆ alkyl, benzyl, or —C(O)OR⁴, wherein R⁴ is C₁ to C₆ alkyl.

In a fourteenth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is

R¹ is —C(O)R^(x);

R² is —C(O)R^(y); and

R^(x) and R^(y) are cycloalkyl independently selected from:

wherein j is 0, 1, 2, or 3;

k is 0, 1, or 2;

n is 0 or an integer selected from 1 though 4;

each R¹³ is independently selected from hydrogen, C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, C₁ to C₆ alkoxy, halogen, hydroxyl, cyano, and nitro; and

R^(a) and R^(b) are independently hydrogen, C₁ to C₆ alkyl, or —C(O)OR⁷, wherein R⁷ is C₁ to C₆ alkyl.

In a fifteenth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is

R¹ is —C(O)R^(x);

R² is —C(O)R^(y); and

R^(x) and R^(y) are independently arylalkyl optionally substituted with (NR^(a)R^(b))alkyl, wherein R^(a) and R^(b) are independently hydrogen, C₁ to C₆ alkyl, or benzyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring selected from and

and

wherein R¹⁵ is hydrogen, C₁ to C₆ alkyl, or benzyl.

In a sixteenth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is and

R¹ and R² are the same and are selected from the group consisting of:

wherein a squiggle bond (

) in the structure indicates that a stereogenic center to which the bond is attached can take either (R)- or (S)-configuration so long as chemical bonding principles are not violated.

In a seventeenth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is

R¹ is —C(O)R^(x);

R² is —C(O)R^(y); and

R^(x) and R^(y) are both t-butoxy.

In an eighteenth embodiment of the first aspect, the present disclosure provides a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein:

Z is oxygen (O), sulfur (S), or NH;

L¹ is

L² is and

R¹ and R² are both hydrogen.

In a nineteenth embodiment of the first aspect, the present disclosure provides a compound, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

-   di-tert-butyl     (2S,2′S)-2,2′-(benzo[1,2-d:4,5-d′]bis[1,3]thiazole-2,6-diylbis(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-5,2-diyl))di(1-pyrrolidinecarboxylate); -   2,6-bis(2-((2S)-2-pyrrolidinyl)-1H-imidazol-5-yl)benzo[1,2-d:4,5-d′]bis[1,3]thiazole; -   methyl     ((1S)-1-(((2S)-2-(4-(6-(2-((2S)-1-((2S)-2-((methoxycarbonyl)amino)-3-methylbutanoyl)-2-pyrrolidinyl)-1H-imidazol-4-yl)[1,3]thiazolo[5,4-f][1,3]benzothiazol-2-yl)-1H-imidazol-2-yl)-1-pyrrolidinyl)carbonyl)-2-methylpropyl)carbamate; -   dimethyl     ([1,3]thiazolo[5,4-f][1,3]benzothiazole-2,6-diylbis(1H-imidazole-4,2-diyl(28)-2,1-pyrrolidinediyl((1R)-2-oxo-1-phenyl-2,1-ethanediyl)))biscarbamate; -   methyl     ((1R)-1-(((2S)-2-(4-(6-(2-((2S)-1-((2R)-2-((methoxycarbonyl)amino)-3-methylbutanoyl)-2-pyrrolidinyl)-1H-imidazol-4-yl)[1,3]thiazolo[5,4-f][1,3]benzothiazol-2-yl)-1H-imidazol-2-yl)-1-pyrrolidinyl)carbonyl)-2-methylpropyl)carbamate;     and

corresponding stereoisomers and tautomers thereof.

In a second aspect the present disclosure provides a composition comprising a compound of Formula (I) or Formula (II):

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, wherein Formula (I) and Formula (II) are defined according to any of the embodiments described above in the first aspect of the present disclosure.

In a first embodiment of the second aspect the composition further comprises at least one additional compound having anti-HCV activity.

In a second embodiment of the second aspect at least one of the additional compounds is an interferon or a ribavirin.

In a third embodiment of the second aspect the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastiod interferon tau.

In a fourth embodiment of the second aspect the present disclosure provides a composition comprising a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and at least one additional compound having anti-HCV activity, wherein at least one of the additional compounds is selected from interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5′-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.

In a fifth embodiment of the second aspect the present disclosure provides a composition comprising a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and at least one additional compound having anti-HCV activity, wherein at least one of the additional compounds is effective to inhibit the function of a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and IMPDH for the treatment of an HCV infection.

In a third aspect the present disclosure provides a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I) or Formula (II):

or a pharmaceutically acceptable salt thereof, wherein Formula (I) and Formula (II) are defined according to any of the embodiments described above in the first aspect of the present disclosure.

In a first embodiment of the third aspect the method further comprises administering at least one additional compound having anti-HCV activity prior to, after or simultaneously with the compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof.

In a second embodiment of the third aspect at least one of the additional compounds is an interferon or a ribavirin.

In a third embodiment of the third aspect the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and lymphoblastoid interferon tau.

In a fourth embodiment of the third aspect the present disclosure provides a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, and at least one additional compound having anti-HCV activity prior to, after or simultaneously with the compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein at least one of the additional compounds is selected from interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5′-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.

In a fifth embodiment of the third aspect the present disclosure provides a method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, and at least one additional compound having anti-HCV activity prior to, after or simultaneously with the compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, wherein at least one of the additional compounds is effective to inhibit the function of a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, and IMPDH for the treatment of an HCV infection.

Other aspects of the present disclosure may include suitable combinations of embodiments disclosed herein.

Yet other aspects and embodiments may be found in the description provided herein.

The description of the present disclosure herein should be construed in congruity with the laws and principals of chemical bonding. In some instances it may be necessary to remove a hydrogen atom in order to accommodate a substituent at any given location.

Certain features of the structure of Formula (I) and Formula (II) are further illustrated below:

In Formula (I) or Formula (II), as depicted above, the “pyrrolidine moiety” on the left side of the “linker” is independent from the “pyrrolidine moiety” on the right side of the linker in respect of, e.g., (1) substituent on the pyrrolidine nitrogen (R¹ or R²), and (2) stereochemistry of the pyrrolidine ring. However, in some circumstances R¹ and R² are preferably the same, and/or the stereochemistry of the two pyrrolidine rings is preferably the same.

It should be understood that the depiction of a pyrrolidine moiety on the “left” side or on the “right” side is for illustration purpose only, which does not in any way limit the scope of the disclosure.

In a pyrrolidine moiety on either side of the molecule, the stereogenic carbon center on the pyrrolidine ring can take either (R)- or (S)-configuration as illustrated below:

Thus, this disclosure is intended to cover all possible stereoisomers even when a single stereoisomer, or no stereochemistry, is described in a structure. The same principle also applies to the R¹ and R² groups.

When Z is NH, the linker group in Formula (I) and the linker group in Formula (II) may be construed to be equivalent, due to the possible tautomerization illustrated below:

In Formula (I) or Formula (II), the linkage between the linker group and L¹ or L² can take place at any available positions on L¹ or L². More specifically, when L¹ or L² is an imidazole group, the linkage can take place at either the C-4 or C-5 position on the imidazole ring. As a person of ordinary skill in the art would understand, due to tautomerization of the imidazole ring, a bonding at the C-4 position may be construed to be equivalent to a bonding at the C-5 position, as illustrated in the following equation:

Likewise, when L¹ or L² is a benzoimidazole

group, the linkage between the linker and the benzoimidazole group can take place at any of the C-4, C-5, C-6, and C-7 positions on the benzene ring of the benzoimidazole group. However, a linkage at the C-5 or C-6 position is preferred. As a person of ordinary skill in the art would understand, due to tautomerization of the imidazole ring, a bonding at the C-5 position may be construed to be equivalent to a bonding at the C-6 position of the benzene ring, as illustrated in the following equation:

Thus, this disclosure is intended to cover all possible tautomers even when a structure depicts only one of them.

In this disclosure, a floating bond (e.g.,

or a floating substituent (e.g., —R¹³) on a structure indicates that the bond or substituent can attach to any available position of the structure by removal of a hydrogen from the available position. It should be understood that in a bicyclic or polycyclic ring structure, unless specifically defined otherwise, the position of a floating bond or a floating substituent does not limit the position of such bond or substituent to a specific ring. Thus, the following two substituents should be construed to be equivalent:

It should be understood that the compounds encompassed by the present disclosure are those that are suitably stable for use as pharmaceutical agent.

It is intended that the definition of any substituent or variable at a particular location in a molecule be independent of its definitions elsewhere in that molecule. For example, for substituent (R¹⁰)_(n), when n is 2, each of the two R¹⁰ groups may be the same or different.

All patents, patent applications, and literature references cited in the specification are herein incorporated by reference in their entirety. In the case of inconsistencies, the present disclosure, including definitions, will prevail.

Definitions

Definitions have been provided above for each of the groups defined. In addition, the following definitions shall be used.

As used herein, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.

Unless stated otherwise, all aryl, cycloalkyl, heteroaryl, and heterocyclyl groups of the present disclosure may be substituted as described in each of their respective definitions. For example, the aryl part of an arylalkyl group may be substituted as described in the definition of the term “aryl.”

The term “acetyl,” as used herein, refers to —C(O)CH₃.

The term “alkenyl,” as used herein, refers to a monovalent, straight or branched hydrocarbon chain having one or more, preferably one to two, double bonds therein. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. Suitable alkenyl groups include, but are not limited to, C₂ to C₁₀ alkenyl groups, such as vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl. An alkenyl group can be unsubstituted or substituted with one or two suitable substituents.

The term “alkoxy,” as used herein, refers to an alkyl group attached to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy group include, but are not limited to, methoxy (CH₃O—), ethoxy (CH₃CH₂O—), and t-butoxy ((CH₃)₃CO—).

The term “alkyl,” as used herein, refers to a group derived from a straight or branched chain saturated hydrocarbon by removal of a hydrogen from one of the saturated carbons. The alkyl group preferably contains from one to ten carbon atoms. Representative examples of alkyl group include, but are not limited to, methyl, ethyl, isopropyl, and tert-butyl.

The term “alkylcarbonyl,” as used herein, refers to an alkyl group attached to the parent molecular moiety through a carbonyl group. Representative examples of alkylcarbonyl group include, but are not limited to, acetyl (—C(O)CH₃), propanoyl (—C(O)CH₂CH₃), n-butyryl (—C(O)CH₂CH₂CH₃), and 2,2-dimethylpropanoyl or pivaloyl (—C(O)C(CH₃)₃).

The term “allyl,” as used herein, refers to the —CH₂CH═CH₂ group.

The term “aryl,” as used herein, refers to a group delved from an aromatic carbocycle by removal of a hydrogen atom from an aromatic ring. The aryl group can be monocyclic, bicyclic or polycyclic, wherein in bicyclic or polycyclic aryl group, the aromatic carbocycle can be fused onto another four- to six-membered aromatic or non-aromatic carbocycle. Representative examples of aryl groups include, but are not limited to, phenyl, indanyl, indenyl, naphthyl, and 1,2,3,4-tetrahydronaphth-5-yl.

The term “arylalkyl,” as used herein, refers to an alkyl group substituted with one, two, or three aryl groups, wherein aryl part of the arylalkyl group may optionally be substituted by one to five substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, C₁ to C₆ alkoxy, halogen, cyano, and nitro groups. Represented examples of arylalkyl include, but are not limited to, benzyl, 2-phenyl-1-ethyl (PhCH₂CH₂—), (naphth-1-yl)methyl, and (naphth-2-yl)methyl.

The term “benzyl,” as used herein, refers to a methyl group on which one of the hydrogen atoms is replaced by a phenyl group, wherein said phenyl group may optionally be substituted by one to five substituents independently selected from methyl, trifluoromethyl (—CF₃), methoxy (—OCH₃), halogen, and nitro (—NO₂). Representative examples of benzyl group include, but are not limited to, PhCH₂—, 4-MeO—C₆H₄—CH₂—, and 2,4,6-tri -methyl-C₆H₄CH₂—.

The term “bridged bicyclic ring,” as used herein, refers to a ring structure comprising a bridgehead between two of the ring members, wherein the ring and the bridgehead optionally may independently comprise one or more, preferably one to two, heteroatoms independently selected from nitrogen, oxygen, and sulfur. Illustrated examples of a bridged bicyclic ring structure include, but are not limited to:

The terms “Cap” and “cap,” as used herein, refer to the group which is placed on the nitrogen atom of the pyrrolidine ring in the compounds of Formula (I) or Formula (II). It should be understood that “Cap” or “cap” can also refer to the reagent which is a precursor to the final “cap” in compounds of Formula (I) or Formula (II) and is used as one of the starting materials in the reaction to append a group on the pyrrolidine nitrogen that results in the final product, a compound which contains the functionalized pyrrolidine that will be present in the compound of Formula (I) or Formula (II).

The term “carbonyl,” as used herein, refers to —C(O)—.

The term “carboxyl,” as used herein, refers to —CO₂H.

The term “cyano,” as used herein, refers to —CN.

The term “cycloalkyl,” as used herein, refers to a group derived from a saturated carbocycle, having preferably three to eight carbon atoms, by removal of a hydrogen atom from the saturated carbocycle, wherein the saturated carbocycle can optionally be fused onto one or two other aromatic or nonaromatic carbocycles. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, and 1,2,3,4-tetrahydronaphth-1-yl.

The term “formyl,” as used herein, refers to —CHO.

The terms “halo” and “halogen,” as used herein, refer to F, Cl, Br, or I.

The term “haloalkoxy,” as used herein, refers to a haloalkyl group attached to the parent molecular moiety through an oxygen atom.

The term “haloalkyl,” as used herein, refers to an alkyl group substituted by at least one halogen atom. The haloalkyl group can be an alkyl group of which all hydrogen atoms are substituted by halogens. Representative examples of haloalkyl include, but are not limited to, trifluoromethyl (CF₃—), 1-chloroethyl (ClCH₂CH₂—), and 2,2,2-trifluoroethyl (CF₃CH₂—).

The term “heteroaryl,” as used herein, refers to group derived from a monocyclic, bicyclic, or polycyclic compound comprising at least one aromatic ring comprising one or more, preferably one to three, heteroatoms independently selected from nitrogen, oxygen, and sulfur, by removal of a hydrogen atom from an aromatic ring thereof. As is well known to those skilled in the art, heteroaryl rings have less aromatic character than their all-carbon counterparts. Thus, for the purposes of the disclosure, a heteroaryl group need only have some degree of aromatic character. Illustrative examples of heteroaryl groups include, but are not limited to, pyridyl, pyridazinyl, pyrimidyl, pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3,)- and (1,2,4)-triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, isoxazolyl, oxazolyl, indolyl, quinolinyl, isoquinolinyl, benzisoxazolyl, benzothiazolyl, benzothienyl, and pyrrolopyridinyl.

The term “heteroarylalkyl,” as used herein, refers to an alkyl group substituted with one, two, or three heteroaryl groups.

The term “heterobicyclyl,” as used herein, refers to a ring structure comprising two fused or bridged rings that include carbon and one or more, preferably one to three, heteroatoms independently selected from nitrogen, oxygen, and sulfur. The heterobicyclic ring structure is a subset of heterocyclic ring and can be saturated or unsaturated. Examples of heterobicyclic ring structures include, but are not limited to, tropane, quinuclidine, and 7-azabicyclo[2.2.1]heptane.

The term “heterocyclyl,” as used herein, refers to a group derived from a monocyclic, bicyclic, or polycyclic compound comprising at least one nonaromatic ring comprising one or more, preferably one to three, heteroatoms independently selected from nitrogen, oxygen, and sulfur, by removal of a hydrogen atom from the nonaromatic ring. The heterocyclyl group encompasses the heterobicyclyl group. The heterocyclyl groups of the present disclosure can be attached to the parent molecular moiety through a carbon atom or a nitrogen atom in the group. Examples of heterocyclyl groups include, but are not limited to, morpholinyl, oxazolidinyl, piperazinyl, piperidinyl, pyrrolidinyl, tetrahydrofuryl, thiomorpholinyl, and indolinyl.

The term “heterocyclylalkyl,” as used herein, refers to an alkyl group substituted with one, two, or three heterocyclyl groups.

The terms “hydroxy” or “hydroxyl,” as used herein, refer to —OH.

The term “nitro,” as used herein, refers to —NO₂.

The term “—NR^(a)R^(b),” as used herein, refers to two groups, R^(a) and R^(b), which are attached to the parent molecular moiety through a nitrogen atom, or alternatively R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a 5- or 6-membered ring or a fused- or bridged-bicyclic ring structure optionally containing one, two, or three additional heteroatom independently selected from nitrogen, oxygen, and sulfur. The term “—NR′R′” is defined similarly.

The term “(NR^(a)R^(b))alkyl,” as used herein, refers to an alkyl group substituted with one, two, or three —NR^(a)R^(b) groups. The term “(NR^(c)R^(d))alkyl” is defined similarly.

The term “oxo,” as used herein, refers to ═O.

The term “sulfonyl,” as used herein, refers to —SO₂—.

Asymmetric centers exist in the compounds of the present disclosure. These centers are designated by the symbols “R” or “S”, depending on the configuration of substituents around the chiral carbon atom. It should be understood that the disclosure encompasses all stereochemical isomeric forms, or mixtures thereof, which possess the ability to inhibit NS5A. Individual stereoisomers of compounds can be prepared synthetically from commercially available starting materials which contain chiral centers or by preparation of mixtures of enantiomeric products followed by separation such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, or direct separation of enantiomers on chiral chromatographic columns. Starting compounds of particular stereochemistry are either commercially available or can be made and resolved by techniques known in the art.

Certain compounds of the present disclosure may also exist in different stable conformational forms which may be separable. Torsional asymmetry due to restricted rotation about an asymmetric single bond, for example because of steric hindrance or ring strain, may permit separation of different conformers. The present disclosure includes each conformational isomer of these compounds and mixtures thereof.

The term “compounds of the present disclosure”, and equivalent expressions, are meant to embrace compounds of Formula (I) or Formula (II), and pharmaceutically acceptable enantiomers, diastereomers, and salts thereof. Similarly, references to intermediates are meant to embrace their salts where the context so permits.

The present disclosure is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium and tritium. Isotopes of carbon include ¹³C and ¹⁴C. Isotopically-labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed. Such compounds may have a variety of potential uses, for example as standards and reagents in determining biological activity. In the case of stable isotopes, such compounds may have the potential to favorably modify biological, pharmacological, or pharmacokinetic properties.

The compounds of the present disclosure can exist as pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt,” as used herein, represents salts or zwitterionic forms of the compounds of the present disclosure which are water or oil-soluble or dispersible, which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, and are effective for their intended use The salts can be prepared during the final isolation and purification of the compounds or separately by reacting a suitable nitrogen atom with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate; digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate, and undecanoate. Examples of acids which can be employed to form pharmaceutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric.

Basic addition salts can be prepared during the final isolation and purification of the compounds by reacting a carboxy group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine. The cations of pharmaceutically acceptable salts include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as nontoxic quaternary amine cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, and N,N′-dibenzylethylenediamine. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.

When it is possible that, for use in therapy, therapeutically effective amounts of a compound of Formula (I) or Formula (II), as well as pharmaceutically acceptable salts thereof, may be administered as the raw chemical, it is possible to present the active ingredient as a pharmaceutical composition. Accordingly, the disclosure further provides pharmaceutical compositions, which include therapeutically effective amounts of compounds of Formula (I) or Formula (II) or pharmaceutically acceptable salts thereof, and one or more, preferably one to three, pharmaceutically acceptable carriers, diluents, or excipients. The term “therapeutically effective amount,” as used herein, refers to the total amount of each active component that is sufficient to show a meaningful patient benefit, e.g., a sustained reduction in viral load. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. The compounds of Formula (I) or Formula (II) and pharmaceutically acceptable salts thereof, are as described above. The carrier(s), diluent(s), or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. In accordance with another aspect of the present disclosure there is also provided a process for the preparation of a pharmaceutical formulation including admixing a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, with one or more, preferably one to three, pharmaceutically acceptable carriers, diluents, or excipients. The term “pharmaceutically acceptable,” as used herein, refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.

Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Dosage levels of between about 0.01 and about 250 milligram per kilogram (“mg/kg”) body weight per day, preferably between about 0.05 and about 100 mg/kg body weight per day of the compounds of the present disclosure are typical in a monotherapy for the prevention and treatment of HCV mediated disease. Typically, the pharmaceutical compositions of this disclosure will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending on the condition being treated, the severity of the condition, the time of administration, the route of administration, the rate of excretion of the compound employed, the duration of treatment, and the age, gender, weight, and condition of the patient. Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient. Generally, treatment is initiated with small dosages substantially less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. In general, the compound is most desirably administered at a concentration level that will generally afford antivirally effective results without causing any harmful or deleterious side effects.

When the compositions of this disclosure comprise a combination of a compound of the present disclosure and one or more additional therapeutic or prophylactic agent, both the compound and the additional agent are usually present at dosage levels of between about 10 to 150%, and more preferably between about 10 and 80% of the dosage normally administered in a monotherapy regimen.

Pharmaceutical formulations may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intracutaneous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, intravenous, or intradermal injections or infusions) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). Oral administration or administration by injection are preferred.

Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil emulsions.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing, and coloring agent can also be present.

Capsules are made by preparing a powder mixture, as described above, and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate, or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, and the like. Lubricants used in these dosage forms include sodium oleate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, betonite, xanthan gum, and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant, and pressing into tablets. A powder mixture is prepared by mixing the compound, suitable comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelating, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or and absorption agent such as betonite, kaolin, or dicalcium phosphate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage, or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, the powder mixture can be run through the tablet machine and the result is imperfectly formed slugs broken into granules. The granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc, or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present disclosure can also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material, and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.

Oral fluids such as solution, syrups, and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners, or saccharin or other artificial sweeteners, and the like can also be added.

Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax, or the like.

The compounds of Formula (I) or Formula (II), and pharmaceutically acceptable salts thereof, can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phopholipids, such as cholesterol, stearylamine, or phophatidylcholines.

The compounds of Formula (I) or Formula (II) and pharmaceutically acceptable salts thereof may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels.

Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research 1986, 3(6), 318.

Pharmaceutical formulations adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.

For treatments of the eye or other external tissues, for example mouth and skin, the formulations are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in oil base.

Pharmaceutical formulations adapted for topical administrations to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical formulations adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.

Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or as enemas.

Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid include a course powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered, dose pressurized aerosols, nebulizers, or insufflators.

Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations.

Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, and soutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

The term “patient” includes both human and other mammals.

The term “treating” refers to: (i) preventing a disease, disorder or condition from occurring in a patient that may be predisposed to the disease, disorder, and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder, or condition, i.e., arresting its development; and (iii) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and/or condition.

The compounds of the present disclosure can also be administered with a cyclosporin, for example, cyclosporin A. Cyclosporin A has been shown to be active against HCV in clinical trials (Hepatology 2003, 38, 1282; Biochem. Biophys. Res. Commun. 2004, 313, 42; J. Gastroenterol. 2003, 38, 567).

Table 1 below lists some illustrative examples of compounds that can be administered with the compounds of this disclosure. The compounds of the disclosure can be administered with other anti-HCV activity compounds in combination therapy, either jointly or separately, or by combining the compounds into a composition.

TABLE 1 Physio- Type of logical Inhibitor or Source Brand Name Class Target Company NIM811 Cyclophilin Novartis Inhibitor Zadaxin Immuno- Sciclone modulator Suvus Methylene blue Bioenvision Actilon TLR9 agonist Coley (CPG10101) Batabulin Anticancer β-tubulin Tularik Inc., (T67) inhibitor South San Francisco, CA ISIS 14803 Antiviral antisense ISIS Pharmaceuticals Inc, Carlsbad, CA/Elan Phamaceuticals Inc., New York, NY Summetrel Antiviral antiviral Endo Pharmaceuticals Holdings Inc., Chadds Ford, PA GS-9132 (ACH- Antiviral HCV Inhibitor Achillion/ 806) Gilead Pyrazolopyrimidine Antiviral HCV Inhibitors Arrow compounds and Therapeutics salts Ltd. From WO- 2005047288 26 May 2005 Levovirin Antiviral IMPDH inhibitor Ribapharm Inc., Costa Mesa, CA Merimepodib Antiviral IMPDH inhibitor Vertex (VX-497) Pharmaceuticals Inc., Cambridge, MA XTL-6865 Antiviral monoclonal XTL (XTL-002) antibody Biopharmaceuticals Ltd., Rehovot, Isreal Telaprevir Antiviral NS3 serine Vertex (VX-950, LY- protease inhibitor Pharmaceuticals 570310) Inc., Cambridge, MA/Eli Lilly and Co. Inc., Indianapolis, IN HCV-796 Antiviral NS5B Replicase Wyeth/ Inhibitor Viropharma NM-283 Antiviral NS5B Replicase Idenix/ Inhibitor Novartis GL-59728 Antiviral NS5B Replicase Gene Labs/ Inhibitor Novartis GL-60667 Antiviral NS5B Replicase Gene Labs/ Inhibitor Novartis 2′C MeA Antiviral NS5B Replicase Gilead Inhibitor PSI 6130 Antiviral NS5B Replicase Roche Inhibitor R1626 Antiviral NS5B Replicase Roche Inhibitor 2′C Methyl Antiviral NS5B Replicase Merck adenosine Inhibitor JTK-003 Antiviral RdRp inhibitor Japan Tobacco Inc., Tokyo, Japan Levovirin Antiviral ribavirin ICN Pharmaceuticals, Costa Mesa, CA Ribavirin Antiviral ribavirin Schering-Plough Corporation, Kenilworth, NJ Viramidine Antiviral Ribavirin Ribapharm Prodrug Inc., Costa Mesa, CA Heptazyme Antiviral ribozyme Ribozyme Pharmaceuticals Inc., Boulder, CO BILN-2061 Antiviral serine protease Boehringer inhibitor Ingelheim Pharma KG, Ingelheim, Germany SCH 503034 Antiviral serine protease Schering inhibitor Plough Zadazim Immune Immune SciClone modulator modulator Pharmaceuticals Inc., San Mateo, CA Ceplene Immuno- immune Maxim modulator modulator Pharmaceuticals Inc., San Diego, CA CellCept Immuno- HCV IgG F. Hoffmann- suppressant immuno- La Roche LTD, suppressant Basel, Switzerland Civacir Immuno- HCV IgG Nabi suppressant immuno- Biopharmaceuticals suppressant Inc., Boca Raton, FL Albuferon - α Interferon albumin IFN-α2b Human Genome Sciences Inc., Rockville, MD Infergen A Interferon IFN InterMune alfacon-1 Pharmaceuticals Inc., Brisbane, CA Omega IFN Interferon IFN-ω Intarcia Therapeutics IFN-β and Interferon IFN-β and Transition EMZ701 EMZ701 Therapeutics Inc., Ontario, Canada Rebif Interferon IFN-β1a Serono, Geneva, Switzerland Roferon A Interferon IFN-α2a F. Hoffmann- La Roche LTD, Basel, Switzerland Intron A Interferon IFN-α2b Schering- Plough Corporation, Kenilworth, NJ Intron A and Interferon IFN-α2b/α1- RegeneRx Zadaxin thymosin Biopharma. Inc., Bethesda, MD/ SciClone Pharmaceuticals Inc, San Mateo, CA Rebetron Interferon IFN- Schering-Plough α2b/ribavirin Corporation, Kenilworth, NJ Actimmune Interferon INF-γ InterMune Inc., Brisbane, CA Interferon-β Interferon Interferon-β-1a Serono Multiferon Interferon Long lasting Viragen/ IFN Valentis Wellferon Interferon Lympho-blastoid GlaxoSmithKline IFN-αn1 plc, Uxbridge, UK Omniferon Interferon natural IFN-α Viragen Inc., Plantation, FL Pegasys Interferon PEGylated IFN- F. Hoffmann- α2a La Roche LTD, Basel, Switzerland Pegasys and Interferon PEGylated IFN- Maxim Ceplene α2a/ Pharmaceuticals immune Inc., San modulator Diego, CA Pegasys and Interferon PEGylated IFN- F. Hoffmann- Ribavirin α2a/ribavirin La Roche LTD, Basel, Switzerland PEG-Intron Interferon PEGylated IFN- Schering-Plough α2b Corporation, Kenilworth, NJ PEG-Intron/ Interferon PEGylated IFN- Schering-Plough Ribavirin α2b/ribavirin Corporation, Kenilworth, NJ IP-501 Liver antifibrotic Indevus protection Pharmaceuticals Inc., Lexington, MA IDN-6556 Liver caspase Idun protection inhibitor Pharmaceuticals Inc., San Diego, CA ITMN-191 Antiviral serine protease InterMune (R-7227) inhibitor Pharmaceuticals Inc., Brisbane, CA GL-59728 Antiviral NS5B Replicase Genelabs Inhibitor ANA-971 Antiviral TLR-7 agonist Anadys Boceprevir Antiviral serine protease Schering inhibitor Plough TMS-435 Antiviral serine protease Tibotec BVBA, inhibitor Mechelen, Belgium BI-201335 Antiviral serine protease Boehringer inhibitor Ingelheim Pharma KG, Ingelheim, Germany MK-7009 Antiviral serine protease Merck inhibitor PF-00868554 Antiviral replicase inhibitor Pfizer ANA598 Antiviral Non-Nucleoside Anadys NS5B Polymerase Pharmaceuticals, Inhibitor Inc., San Diego, CA, USA IDX375 Antiviral Non-Nucleoside Idenix Replicase Pharmaceuticals, Inhibitor Cambridge, MA, USA BILB 1941 Antiviral NS5B Polymerase Boehringer Inhibitor Ingelheim Canada Ltd R&D, Laval, QC, Canada PSI-7851 Antiviral Nucleoside Pharmasset, Polymerase Princeton, inhibitor NJ, USA VCH-759 Antiviral NS5B Polymerase ViroChem Inhibitor Pharma VCH-916 Antiviral NS5B Polymerase ViroChem Inhibitor Pharma GS-9190 Antiviral NS5B Polymerase Gilead Inhibitor Peg- Antiviral Interferon ZymoGenetics/ interferon Bristol-Myers lamda Squibb

The compounds of the present disclosure may also be used as laboratory reagents. Compounds may be instrumental in providing research tools for designing of viral replication assays, validation of animal assay systems and structural biology studies to further enhance knowledge of the HCV disease mechanisms. Further, the compounds of the present disclosure are useful in establishing or determining the binding site of other antiviral compounds, for example, by competitive inhibition.

The compounds of this disclosure may also be used to treat or prevent viral contamination of materials and therefore reduce the risk of viral infection of laboratory or medical personnel or patients who come in contact with such materials, e.g., blood, tissue, surgical instruments and garments, laboratory instruments and garments, and blood collection or transfusion apparatuses and materials.

This disclosure is intended to encompass compounds having Formula (I) or Formula (II) when prepared by synthetic processes or by metabolic processes including those occurring in the human or animal body (in vivo) or processes occurring in vitro.

The abbreviations used in the present application, including particularly in the illustrative examples which follow, are well-known to those skilled in the art. Some of the abbreviations used are as follows:

-   Me methyl; -   Et ethyl; -   t-Bu tert-butyl; -   iPr isopropyl; -   min minutes; -   rt or RT room temperature or retention time (context will dictate); -   TFA trifluoroacetic acid; -   h or hr hours; -   DMSO dimethylsulfoxide; -   DME dimethyl ether; -   LDA Lithium diisopropylamide; -   NBS N-Bromosuccinimide; -   SEM-Cl 2-(Trimethylsilyl)ethoxymethyl chloride; -   TBAF tetrabutylammonium fluoride; -   EEDQ 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; -   HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium     hexafluorophosphate; -   iPr₂EtN diisopropylethylamine; -   DIEA diisopropylethylamine; -   DIPEA diisopropylethylamine; -   Hunig's diisopropylethylamine; -   Boc or BOC tert-butoxycarbonyl; -   DMAP 4-dimethylaminopyridine; -   HCl hydrochloric acid; -   Na₂SO₄ sodium sulfate; -   MgSO₄ magnesium sulfate, -   PdCl₂(PPh₃)₂

bis(triphenylphosphine)palladium(II) dichloride; MCX cartridge Waters Oasis® MCX LP extraction cartridge.

The present disclosure will now be described in connection with certain embodiments which are not intended to limit its scope. On the contrary, the present disclosure covers all alternatives, modifications, and equivalents as can be included within the scope of the claims. Thus, the following examples, which include specific embodiments, will illustrate one practice of the present disclosure, it being understood that the examples are for the purposes of illustration of certain embodiments and are presented to provide what is believed to be the most useful and readily understood description of its procedures and conceptual aspects.

Starting materials can be obtained from commercial sources or prepared by well-established literature methods known to those of ordinary skill in the art.

EXAMPLES AND METHOD OF PREPARATION

Purity assessment and low resolution mass analysis were conducted on a Shimadzu LC system coupled with Waters Micromass ZQ MS system.

Examples for the present disclosure could be prepared according to the following synthetic schemes.

Scheme 1 depicts that 4-methylhydroxy imidazoles could be prepared by the reaction of imidamides with 1,3-dihydroxyacetone in refluxing ammonium hydroxide. The primary alcohol could then be oxidized to an aldehyde or to a carboxylic acid or ester using known reagents or processes.

Scheme 2 depicts an alternative method for preparing imidazoles with a 4-carboxaldehyde, 4-carboxylic acid or 4-carboxylic ester. Lithium halogen exchange of know 4-halo bromo or iodo)imidazoles with alkyl lithium could form a nucleophile which could be quenched with the appropriate electrophile such as (but not limited to) DMF, carbon dioxide or methyl chloroformate. Reaction of the functionalized imidazole with 1,4-diol-2,5-diaminobenzene, 1,4-dithiol-2,5-diaminobenzene or 1,2,4,5-tertaminobenzene could form C₂-symmetric bisbenzoxazoles, C₂-symmetric bisbenzothiazoles or bisbenzimidazoles with two attached 4-imidazoles. These compounds could form from the condensation of an aldehyde with the aniline nitrogens to from imines followed by cyclization and spontaneous or directed oxidation or by first preparing the amide from the 4-imidazole carboxylic acid and the aniline nitrogens and then cyclizing under acidic conditions at elevated temperatures.

Scheme 3 is similar to Scheme 2, but depicts the formation of meso-bisbenzoxazoles or meso-bisbenzothiazoles with two attached 4-imidazoles from 1,3-diol or 1,3-dithiol-4,6-diaminobenzene.

Scheme 4 depicts that benzimidazole 6-carboxylic acid could be formed by the condensation of methyl 3,4-diaminobenzoate with carboxylic acids (such as praline) followed by acid catalyzed cyclization and hydrolysis of the carboxylic ester. The benzimidazole 6-carboxylic acid could then be used to form C₂-symmetric bisbenzoxazoles, C₂-symmetric bisbenzothiazoles or bisbenzimidazoles with two attached 5-benzimidazoles. This could be accomplished by standard amide coupling followed by acid catalyzed bis-cyclization.

Scheme 5 is similar to Scheme 4, but depicts the formation of meso-bisbenzoxazoles or bisbenzothiazoles with two attached 5-benzimidazoles from 1,3-diol-4,6-diaminobenzene or 1,3-dithiol-4,6-diaminobenzene.

Scheme 6 depicts a method for preparing C₂-symmetric bisbenzoxazoles, C₂-symmetric bisbenzothiazoles or bisbenzimidazoles with one 4-imidazole and one 5-benzimidazole. This could be accomplished by the reaction of one equivalent of a 4-functionalized imidazole (carboxylic acid or carboxaldehyde) with one equivalent of 1,3-diol-4,6-diaminobenzene, 1,3-dithiol-4,6-diaminobenzene or 1,2,4,5-tertaminobenzene to form an amide or a mono-benzoxazole, benzothiazole or benzimidazole, followed by standard amid coupling with a benzimidazole 6-carboxylic acid and acid catalyzed mono- or bis-cyclization.

Scheme 7 is similar to Scheme 2, but depicts the formation of meso-bisbenzoxazoles or meso-bisbenzothiazoles with one 4-imidazole and one 5-benzimidazole.

Scheme 8 depicts the final synthetic steps for preparing molecules in this disclosure. First, standard deprotection of the molecules which could be synthesized in schemes 2-7 (TFA or HCl, if PG were a BOC group for example) followed by standard amide coupling (for example HATU, DEPBT, EDC or CDI) with a carboxylic acid.

The examples for the present disclosure were prepared according to the following synthetic scheme.

Intermediate 1 (S)-tert-Butyl 2-(5-formyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)pyrrolidine-1-carboxylate

n-Butyl lithium (2.5 M) in hexanes (1.6 mL, 4.00 mmol) was added dropwise to a stirring solution of (S)-tert-butyl 2-(5-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)pyrrolidine-1-carboxylate (prepared by methods described in WO 2008/021928, WO 2008/021936 and WO 2008/021927)(1.70 g, 3.81 mmol) in tetrahydrofuran (20 mL) at −78° C. The reaction mixture was stirred at −78° C. for 7 min, and then quenched with dimethylformamide (0.59 mL, 7.6 mmol) in one portion. The mixture was warmed to room temperature by removing the cooling bath, allowed to stir for 1 h, diluted with water and extracted with ethyl acetate (2×). The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (0 to 100% ethyl acetate in hexanes) to afford (S)-tert-butyl 2-(5-formyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)pyrrolidine-1-carboxylate (500 mg, 1.26 mmol, 33% yield) as a yellow oil and a 2:3 mixture of regioisomers.

LC-MS retention time 2.09 min; m/z 396 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Waters Sunfire 5u C18 4.6×50 mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min, a gradient of 100% solvent A/0% solvent B to 0% solvent A/100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 10% acetonitrile/90% water/0.1% TFA and solvent B was 90% acetonitrile/10% water/0.1% TFA. MS data was determined using a Micromass Platform for LC in electrospray mode. ¹H NMR (500 MHz, CDCl₃) δ ppm 9.85 (br s, 1H), 7.59 (s, 1H), 5.91 (d, J=10.7 Hz, 0.6H), 5.46 (d, J=10.7 Hz, 0.4H), 5.21 (d, J=11.0 Hz, 1H), 5.10-4.86 (m, 1H), 3.76-3.43 (m, 4H), 2.49-1.87 (m, 4H), 1.40 (br s, 6H), 1.22 (br s, 3H), 0.99-0.83 (m, 2H), 0.00 (s, 9H).

Intermediate 2 Di-tert-butyl (2S,2′S)-2,2′-(benzo[1,2-d:4,5-d′]bis[1,3]thiazole-2,6-diylbis(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-5,2-diyl))di(1-pyrrolidinecarboxylate)

A stirred solution of (S)-tert-butyl 2-(5-formyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)pyrrolidine-1-carboxylate (50 mg, 0.13 mmol) and 2,5-diaminobenzene-1,4-dithiol dihydrochloride (62.0 mg, 0.25 mmol) in dimethyl sulfoxide (1 mL) was heated to 120° C. for 3 h. The reaction mixture was cooled and concentrated to afford a crude black tar containing di-tert-butyl (2S,2′S)-2,2-f-(benzo[1,2-d:4,5-d′]bis[1,3]thiazole-2,6-diylbis(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-5,2-diyl))di(1-pyrrolidinecarboxylate) which was used without purification.

Intermediate 3 2,6-Bis(2-((2S)-2-pyrrolidinyl)-1H-imidazol-5-yl)benzo[1,2-d:4,5-d′]bis[1,3]thiazole tetratrifluoroacetate

TFA (4.0 mL, 52 mmol) was added to crude di-tert-butyl (2S,2′S)-2,21-(benzo[1,2-d:4,5-d′]bis[1,3]thiazole-2,6-diylbis(1-((2-(trimethylsilyl)ethoxy)methyl)-1,4-imidazole-5,2-diyl))di(1-pyrrolidinecarboxylate) suspended in dichloromethane (4 mL). The solution immediately became clear and was stirred at room temperature for 3 h. The reaction mixture was concentrated to dryness in vacuo, and purified by preparative HPLC (acetonitrile/water/0.1% TFA) to yield 2,6-bis(2-((2S)-2-pyrrolidinyl)-1H-imidazol-5-yl)benzo[1,2-d:4,5-d′]bis[1,3]thiazole tetratrifluoroacetate (40 mg, 0.044 mmol, 34% yield) as a yellow oil.

LC-MS retention time 0.78 min; m/z 485.1 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Waters Sunfire 5u C18 4.6×50 mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min, a gradient of 100% solvent A/0% solvent B to 0% solvent A/100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 10% acetonitrile/90% water/0.1% TFA and solvent B was 90% acetonitrile/10% water/0.1% TFA. MS data was determined using a Micromass Platform for LC in electrospray mode.

Example 1 Methyl ((1S)-1-(((2S)-2-(4-(6-(2-((28)-1-((2S)-2-((methoxycarbonyl)amino)-3-methylbutanoyl)-2-pyrrolidinyl)-1H-imidazol-4-yl)[1,3]thiazolo[5,4-f][1,3]benzothiazol-2-yl)-1H-imidazol-2-yl)-1-pyrrolidinyl)carbonyl)-2-methylpropyl)carbamate, bistrifluoracetate

HATU (39.7 mg, 0.104 mmol) was added to a stirred solution of 2,6-bis(2-((2S)-2-pyrrolidinyl)-1H-imidazol-5-yl)benzo[1,2-d:4,5-d′]bis[1,3]thiazole (40 mg, 0.044 mmol), (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid (19.1 mg, 0.109 mmol) and DIEA (0.076 mL, 0.44 mmol) in dimethylformamide (2 mL). The reaction was stirred for 4 h at room temperature (incomplete by LCMS) and additional (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid (19.1 mg, 0.109 mmol), DIEA (0.076 mL, 0.44 mmol) and HATU (39.7 mg, 0.104 mmol) were added and the mixture was stirred overnight at room temperature. The reaction mixture was diluted with methanol (2 mL) and water (2 mL), stirred for 15 min at room temperature and concentrated to dryness in vacuo. The residue was purified by preparative HPLC (acetonitrile/water/0.1% TFA) to afford methyl ((1S)-1-(((2S)-2-(4-(6-(2-((2S)-1-((2S)-2-((methoxycarbonyl)amino)-3-methylbutanoyl)-2-pyrrolidinyl)-1H′-imidazol-4-yl)[1,3]thiazolo[5,4-f][1,3]benzothiazol-2-yl)-1H-imidazol-2-yl)-1-pyrrolidinyl)carbonyl)-2-methylpropyl)carbamate bisitrifluoroacetate (3.3 mg, 3.3 μmol, 7.5% yield) as a yellow oil.

LC-MS retention time 1.68 min; m/z 777.4 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna 10u C18 4.6×50 mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 5 mL/min, a gradient of 100% solvent A/0% solvent B to 0% solvent A/100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 5% acetonitrile/95% water/10 mM ammonium acetate and solvent B was 5% water/95% acetonitrile/10 mM ammonium acetate. MS data was determined using a Micromass Platform for LC in electrospray mode. ¹H NMR (500 MHz, MeOD-d₄) δ ppm 8.60-8.54 (m, 2H), 8.06-7.99 (m, 2H), 5.23 (dd, J=7.6, 6.1 Hz, 2H), 4.25 (d, J=7.3 Hz, 2H), 4.10-4.03 (m, 2H), 3.90 (dt, J=10.0, 7.1 Hz, 2H), 3.66 (s, 6H), 2.50-2.40 (m, 2H), 2.35-2.00 (m, 8H), 1.01-0.95 (m, 12H).

Example 2 Dimethyl ([1,3]thiazolo[5,4-f][1,3]benzothiazole-2,6-diylbis(1H-imidazole-4,2-diyl(2S)-2,1-pyrrolidinediyl((1R)-2-oxo-1-phenyl-2,1-ethanediyl)))biscarbamate, bistrifluoracetate

HATU (43.6 mg, 0.115 mmol) was added to a stirred solution of 2,6-bis(2-((2S)-2-pyrrolidinyl)-1H-imidazol-5-yl)benzo[1,2-d:4,5-d′]bis[1,3]thiazole (27 mg, 0.029 mmol), (R)-2-(methoxycarbonylamino)-2-phenylacetic acid (24.6 mg, 0.118 mmol) and DIEA (0.051 mL, 0.29 mmol) in dimethylformamide (2 mL). The reaction was stirred for 16 h at room temperature. The reaction mixture was diluted with methanol (2 mL) and water (2 mL), stirred for 15 min at room temperature and concentrated to dryness in vacuo. The residue was purified by preparative HPLC (acetonitrile/water/0.1% TFA) to afford dimethyl ([1,3]thiazolo[5,4-f][1,3]benzothiazole-2,6-diylbis(1H′-imidazole-4,2-diyl(2S)-2,1-pyrrolidinediyl((1R)-2-oxo-1-phenyl-2,1-ethanediyl)))biscarbamate, bistrifluoracetate (14.9 mg, 0.014 mmol, 47.4% yield) as a pale yellow solid.

LC-MS retention time 1.41 min; m/z 867.2 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Waters Sunfire 5u C18 4.6×50 mm column using a SPD-10AV UV-V is detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min, a gradient of 100% solvent A/0% solvent B to 0% solvent A/100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 10% acetonitrile/90% water/0.1% TFA and solvent B was 90% acetonitrile/10% water/0.1% TFA. MS data was determined using a Micromass Platform for LC in electrospray mode. ¹H NMR (500 MHz, MeOD-d₄) δ ppm 8.46-8.60 (m, 2H), 7.32-7.52 (m, 10H), 7.00-7.10 (m, 2H), 5.55 (s, 2H), 5.27 (dd, J=8.2, 3.4 Hz, 2H), 4.03 (t, J=10.8 Hz, 2H), 3.58-3.78 (m, 8H), 2.24-2.41 (m, 2H), 2.10-2.24 (m, 4H), 1.84-2.01 (m, 2H).

Example 3 Methyl ((1R)-1-(((2S)-2-(4-(6-(2-((2S)-1-((2R)-2-((methoxycarbonyl)amino)-3-methylbutanoyl)-2-pyrrolidinyl)-1H-imidazol-4-yl)[1,3]thiazolo[5,4-f][1,3]benzothiazol-2-yl)-1H-imidazol-2-yl)-1-pyrrolidinyl)carbonyl)-2-methylpropyl)carbamate, bistrifluoracetate

HATU (43.6 mg, 0.115 mmol) was added to a stirred solution of 2,6-bis(2-((2S)-2-pyrrolidinyl)-1H-imidazol-5-yl)benzo[1,2-d:4,5-d′]bis[1,3]thiazole (27 mg, 0.029 mmol), (R)-2-(methoxycarbonylamino)-3-methylbutanoic acid (20.6 mg, 0.118 mmol) and DIEA (0.051 mL, 0.29 mmol) in dimethylformamide (2 mL). The reaction was stirred overnight at room temperature. The reaction mixture was diluted with methanol (2 mL) and water (2 mL), stirred for 15 min at room temperature and concentrated to dryness in vacuo. The residue was purified by preparative HPLC (acetonitrile/water/0.1% TFA) to afford methyl ((1R)-1-(((2S)-2-(4-(6-(2-((2S)-1-((2R)-2-((methoxycarbonyl)amino)-3-methylbutanoyl)-2-pyrrolidinyl)-1H-imidazol-4-yl)[1,3]thiazolo[5,4-f][1,3]benzothiazol-2-yl)-1H-imidazol-2-yl)-1-pyrrolidinyl)carbonyl)-2-methylpropyl)carbamate bistrifluoracetate (13.0 mg, 0.013 mmol, 44.0% yield) as a pale yellow solid.

LC-MS retention time 1.30 min; m/z 777.2 (MH+). LC data was recorded on a Shimadzu LC-10AS liquid chromatograph equipped with a Waters Sunfire 5u C18 4.6×50 mm column using a SPD-10AV UV-Vis detector at a detector wave length of 220 nM. The elution conditions employed a flow rate of 4 mL/min, a gradient of 100% solvent A/0% solvent B to 0% solvent A/100% solvent B, a gradient time of 3 min, a hold time of 1 min, and an analysis time of 4 min where solvent A was 10% acetonitrile/90% water/0.1% TFA and solvent B was 90% acetonitrile/10% water/0.1% TFA. MS data was determined using a Micromass Platform for LC in electrospray mode. ¹H NMR (500 MHz, MeOD-d₄) δ ppm 8.64-8.55 (m, 2H), 8.15-7.95 (m, 2H), 5.29 (dd, J=8.1, 3.5 Hz, 2H), 4.27 (d, J=7.3 Hz, 2H), 4.16-4.04 (m, 2H), 3.87-3.79 (m, 2H), 3.68 (s, 6H), 2.53-2.39 (m, 2H), 2.31-2.01 (m, 8H), 1.07-0.82 (m, 12H).

Synthesis of Common Caps

Additional LC conditions applicable to the current section, unless noted otherwise.

Cond.—MS-W1

-   Column=XTERRA 3.0×50 mm S7 -   Start % B=0 -   Final % B=100 -   Gradient time=2 min -   Stop time=3 min -   Flow Rate=5 mL/min -   Wavelength=220 nm -   Solvent A=0.1% TFA in 10% methanol/90% H₂O -   Solvent B=0.1% TFA in 90% methanol/10% H₂O

Cond.—MS-W2

-   Column=XTERRA 3.0×50 mm S7 -   Start % B=0 -   Final % B=100 -   Gradient time=3 min -   Stop time=4 min -   Flow Rate=4 mL/min -   Wavelength=220 nm -   Solvent A=0.1% TFA in 10% methanol/90% H2O -   Solvent B=0.1% TFA in 90% methanol/10% H2O

Cond.—MS-W5

-   Column=XTERRA 3.0×50 mm S7 -   Start % B=0 -   Final % B=30 -   Gradient time=2 min -   Stop time=3 min -   Flow Rate=5 mL/min -   Wavelength=220 nm -   Solvent A=0.1% TFA in 10% methanol/90% H2O -   Solvent B=0.1% TFA in 90% methanol/10% H2O

Cond.—D1

-   Column=XTERRA C18 3.0×50 mm S7 -   Start % B=0 -   Final % B=100 -   Gradient time=3 min -   Stop time=4 min -   Flow Rate=4 mL/min -   Wavelength=220 nm -   Solvent A=0.1% TFA in 10% methanol/90% H2O -   Solvent B=0.1% TFA in 90% methanol/10% H2O

Cond.—D2

-   Column=Phenomenex-Luna 4.6×50 mm S10 -   Start % B=0 -   Final % B=100 -   Gradient time=3 min -   Stop time=4 min -   Flow Rate=4 mL/min -   Wavelength=220 nm -   Solvent A=0.1% TFA in 10% methanol/90% H2O -   Solvent B=0.1% TFA in 90% methanol/10% H2O

Cond.—M3

-   Column=XTERRA C18 3.0×50 mm S7 -   Start % B=0 -   Final % B=40 -   Gradient time=2 min -   Stop time=3 min -   Flow Rate=5 mL/min -   Wavelength=220 nm -   Solvent A=0.1% TFA in 10% methanol/90% H2O -   Solvent B=0.1% TFA in 90% methanol/10% H2O

Condition I

-   Column=Phenomenex-Luna 3.0×50 mm S10 -   Start % B=0 -   Final % B=100 -   Gradient time=2 min -   Stop time=3 min -   Flow Rate=4 mL/min -   Wavelength=220 nm -   Solvent A=0.1% TFA in 10% methanol/90% H2O -   Solvent B=0.1% TFA in 90% methanol/10% H2O

Condition II

-   Column=Phenomenex-Luna 4.6×50 mm S10 -   Start % B=0 -   Final % B=100 -   Gradient time=2 min -   Stop time=3 min -   Flow Rate=5 mL/min -   Wavelength=220 nm -   Solvent A=0.1% TFA in 10% methanol/90% H2O -   Solvent B=0.1% TFA in 90% methanol/10% H2O

Condition III

-   Column=XTERRA C18 3.0×50 mm S7 -   Start % B=0 -   Final % B=100 -   Gradient time=3 min -   Stop time=4 min -   Flow Rate=4 mL/min -   Wavelength=220 nm -   Solvent A=0.1% TFA in 10% methanol/90% H2O -   Solvent B=0.1% TFA in 90% methanol/10% H2O

A suspension of 10% Pd/C (2.0 g) in methanol (10 mL) was added to a mixture of (R)-2-phenylglycine (10 g, 66.2 mmol), formaldehyde (33 mL of 37% wt. in water), 1N HCl (30 mL) and methanol (30 mL), and exposed to H₂ (60 psi) for 3 hours. The reaction mixture was filtered through diatomaceous earth (Celite®), and the filtrate was concentrated in vacuo. The resulting crude material was recrystallized from isopropanol to provide the HCl salt of Cap-1 as a white needle (4.0 g). Optical rotation: −117.1° [c=9.95 mg/mL in H₂O; λ=589 nm]. ¹H NMR (DMSO-d₆, δ=2.5 ppm, 500 MHz): δ 7.43-7.34 (m, 5H), 4.14 (s, 1H), 2.43 (s, 6H); LC (Cond. I): RT=0.25; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₀H₁₄NO₂ 180.10; found 180.17; HRMS: Anal. Calcd. for [M+H]⁺ C₁₀H₁₄NO₂ 180.1025; found 180.1017.

NaBH₃CN (6.22 g, 94 mmol) was added in portions over a few minutes to a cooled (ice/water) mixture of (R)-2-Phenylglycine (6.02 g, 39.8 mmol) and methanol (100 mL), and stirred for 5 minutes. Acetaldehyde (10 mL) was added dropwise over 10 minutes and stirring was continued at the same cooled temperature for 45 minutes and at ambient temperature for ˜6.5 hours. The reaction mixture was cooled back with ice-water bath, treated with water (3 mL) and then quenched with a dropwise addition of concentrated HCl over ˜45 minutes until the pH of the mixture was ˜1.5-2.0. The cooling bath was removed and the stirring was continued while adding concentrated HCl in order to maintain the pH of the mixture around 1.5-2.0. The reaction mixture was stirred overnight, filtered to remove the white suspension, and the filtrate was concentrated in vacuo. The crude material was recrystallized from ethanol to afford the HCl salt of Cap-2 as a shining white solid in two crops (crop-1: 4.16 g; crop-2: 2.19 g). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): 10.44 (1.00, br s, 1H), 7.66 (m, 2H), 7.51 (m, 3H), 5.30 (s, 1H), 3.15 (br m, 2H), 2.98 (br m, 2H), 1.20 (app br s, 6H). Crop-1: [α]²⁵ −102.21° (c=0.357, H₂O); crop-2: [α]²⁵ −99.7° (c=0.357, H₂O). LC (Cond. I): RT=0.43 min; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₂H₁₈NO₂: 208.13; found 208.26.

Acetaldehyde (5.0 mL, 89.1 mmol) and a suspension of 10% Pd/C (720 mg) in methanol/H₂O (4 mL/1 mL) was sequentially added to a cooled (˜15° C.) mixture of (R)-2-phenylglycine (3.096 g, 20.48 mmol), 1N HCl (30 mL) and methanol (40 mL). The cooling bath was removed and the reaction mixture was stirred under a balloon of H₂ for 17 hours. An additional acetaldehyde (10 mL, 178.2 mmol) was added and stirring continued under H₂ atmosphere for 24 hours [Note: the supply of H₂ was replenished as needed throughout the reaction]. The reaction mixture was filtered through diatomaceous earth (Celite®), and the filtrate was concentrated in vacuo. The resulting crude material was recrystallized from isopropanol to provide the HCl salt of (R)-2-(ethylamino)-2-phenylacetic acid as a shining white solid (2.846 g). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): δ 14.15 (br s, 1H), 9.55 (br s, 2H), 7.55-7.48 (m, 5H), 2.88 (br m, 1H), 2.73 (br m, 1H), 1.20 (app t, J=7.2, 3H). LC (Cond. I): RT=0.39 min; >95% homogeneity index; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₀H₁₄NO₂: 180.10; found 180.18.

A suspension of 10% Pd/C (536 mg) in methanol/H₂O (3 mL/1 mL) was added to a mixture of (R)-2-(ethylamino)-2-phenylacetic acid/HCl (1.492 g, 6.918 mmol), formaldehyde (20 mL of 37% wt. in water), 1N HCl (20 mL) and methanol (23 mL). The reaction mixture was stirred under a balloon of H₂ for ˜72 hours, where the H₂ supply was replenished as needed. The reaction mixture was filtered through diatomaceous earth (Celite®) and the filtrate was concentrated in vacuo. The resulting crude material was recrystallized from isopropanol (50 mL) to provide the HCl salt of Cap-3 as a white solid (985 mg). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): δ 10.48 (br s, 1H), 7.59-7.51 (m, 5H), 5.26 (s, 1H), 3.08 (app br s, 2H), 2.65 (br s, 3H), 1.24 (br m, 3H). LC (Cond. I): RT=0.39 min; >95% homogeneity index; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₁H₁₆NO₂: 194.12; found 194.18; HRMS: Anal. Calcd. for [M+H]⁺ C₁₁H₁₆NO₂: 194.1180; found 194.1181.

ClCO₂Me (3.2 mL, 41.4 mmol) was added dropwise to a cooled (ice/water) THF (410 mL) semi-solution of (R)-tert-butyl 2-amino-2-phenylacetate/HCl (9.877 g, 40.52 mmol) and diisopropylethylamine (14.2 mL, 81.52 mmol) over 6 min, and stirred at similar temperature for 5.5 hours. The volatile component was removed in vacuo, and the residue was partitioned between water (100 mL) and ethyl acetate (200 mL). The organic layer was washed with 1N HCl (25 mL) and saturated NaHCO₃ solution (30 mL), dried (MgSO₄), filtered, and concentrated in vacuo. The resultant colorless oil was triturated from hexanes, filtered and washed with hexanes (100 mL) to provide (R)-tert-butyl 2-(methoxycarbonylamino)-2-phenylacetate as a white solid (7.7 g). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): 7.98 (d, J=8.0, 1H), 7.37-7.29 (m, 5H), 5.09 (d, J=8, 1H), 3.56 (s, 3H), 1.33 (s, 9H). LC (Cond. I): RT=1.53 min; ˜90% homogeneity index; LC/MS: Anal. Calcd. for [M+Na]⁺ C₁₄H₁₉NNaO₄: 288.12; found 288.15.

TFA (16 mL) was added dropwise to a cooled (ice/water) CH₂Cl₂ (160 mL) solution of the above product over 7 minutes, and the cooling bath was removed and the reaction mixture was stirred for 20 hours. Since the deprotection was still not complete, an additional TFA (1.0 mL) was added and stirring continued for an additional 2 hours. The volatile component was removed in vacuo, and the resulting oil residue was treated with diethyl ether (15 mL) and hexanes (12 mL) to provide a precipitate. The precipitate was filtered and washed with diethyl ether/hexanes (˜1:3 ratio; 30 mL) and dried in vacuo to provide Cap-4 as a fluffy white solid (5.57 g). Optical rotation: −176.9° [c=3.7 mg/mL in H₂O; λ=589 nm]. ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): δ 12.84 (br s, 1H), 7.96 (d, J=8.3, 1H), 7.41-7.29 (m, 5H), 5.14 (d, J=8.3, 1H), 3.55 (s, 3H). LC (Cond. I): RT=1.01 min; >95% homogeneity index; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₀H₁₂NO₄ 210.08; found 210.17; HRMS: Anal. Calcd. for [M+H]⁺ C₁₀H₁₂NO₄ 210.0766; found 210.0756.

A mixture of (R)-2-phenylglycine (1.0 g, 6.62 mmol), 1,4-dibromobutane (1.57 g, 7.27 mmol) and Na₂CO₃ (2.10 g, 19.8 mmol) in ethanol (40 mL) was heated at 100° C. for 21 hours. The reaction mixture was cooled to ambient temperature and filtered, and the filtrate was concentrated in vacuo. The residue was dissolved in ethanol and acidified with 1N HCl to pH 3-4, and the volatile component was removed in vacuo. The resulting crude material was purified by a reverse phase HPLC (water/methanol/TFA) to provide the TFA salt of Cap-5 as a semi-viscous white foam (1.0 g). ¹H NMR (DMSO-d₆, δ=2.5, 500 MHz) δ 10.68 (br s, 1H), 7.51 (m, 5H), 5.23 (s, 1H), 3.34 (app br s, 2H), 3.05 (app br s, 2H), 1.95 (app br s, 4H); RT=0.30 minutes (Cond. I); >98% homogeneity index; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₂H₁₆NO₂: 206.12; found 206.25.

The TFA salt of Cap-6 was synthesized from (R)-2-phenylglycine and 1-bromo-2-(2-bromoethoxy)ethane by using the method of preparation of Cap-5. ¹H NMR (DMSO-d₆, δ=2.5, 500 MHz) δ 12.20 (br s, 1H), 7.50 (m, 5H), 4.92 (s, 1H), 3.78 (app br s, 4H), 3.08 (app br s, 2H), 2.81 (app br s, 2H); RT=0.32 minutes (Cond. I); >98% LC/MS: Anal. Calcd. for [M+H]⁺ C₁₂H₁₆NO₃: 222.11; found 222.20; HRMS: Anal. Calcd. for [M+H]⁺ C₁₂H₁₆NO₃: 222.1130; found 222.1121.

A CH₂Cl₂ (200 mL) solution of p-toluenesulfonyl chloride (8.65 g, 45.4 mmol) was added dropwise to a cooled (−5° C.) CH₂Cl₂ (200 mL) solution of (S)-benzyl 2-hydroxy-2-phenylacetate (10.0 g, 41.3 mmol), triethylamine (5.75 mL, 41.3 mmol) and 4-dimethylaminopyridine (0.504 g, 4.13 mmol), while maintaining the temperature between −5° C. and 0° C. The reaction was stirred at 0° C. for 9 hours, and then stored in a freezer (−25° C.) for 14 hours. It was allowed to thaw to ambient temperature and washed with water (200 mL), 1N HCl (100 mL) and brine (100 mL), dried (MgSO₄), filtered, and concentrated in vacuo to provide benzyl 2-phenyl-2-(tosyloxy)acetate as a viscous oil which solidified upon standing (16.5 g). The chiral integrity of the product was not checked and that product was used for the next step without further purification. ¹H NMR (DMSO-d₆, δ=2.5, 500 MHz) δ 7.78 (d, J=8.6, 2H), 7.43-7.29 (m, 10H), 7.20 (m, 2H), 6.12 (s, 1H), 5.16 (d, J=12.5, 1H), 5.10 (d, J=12.5, 1H), 2.39 (s, 3H). RT=3.00 (Cond. III); >90% homogeneity index; LC/MS: Anal. Calcd. for [M+H]⁺ C₂₂H₂₀NaO₅S: 419.09; found 419.04.

A THF (75 mL) solution of benzyl 2-phenyl-2-(tosyloxy)acetate (6.0 g, 15.1 mmol), 1-methylpiperazine (3.36 mL, 30.3 mmol) and N,N-diisopropylethylamine (13.2 mL, 75.8 mmol) was heated at 65° C. for 7 hours. The reaction was allowed to cool to ambient temperature and the volatile component was removed in vacuo. The residue was partitioned between ethylacetate and water, and the organic layer was washed with water and brine, dried (MgSO₄), filtered, and concentrated in vacuo. The resulting crude material was purified by flash chromatography (silica gel, ethyl acetate) to provide benzyl 2-(4-methylpiperazin-1-yl)-2-phenylacetate as an orangish-brown viscous oil (4.56 g). Chiral HPLC analysis (Chiralcel OD-H) indicated that the sample is a mixture of enantiomers in a 38.2 to 58.7 ratio. The separation of the enantiomers were effected as follow: the product was dissolved in 120 mL of ethanol/heptane (1:1) and injected (5 mL/injection) on chiral HPLC column (Chiracel OJ, 5 cm ID×50 cm L, 20 μm) eluting with 85:15 Heptane/ethanol at 75 mL/min, and monitored at 220 nm. Enantiomer-1 (1.474 g) and enantiomer-2 (2.2149 g) were retrieved as viscous oil. ¹H NMR (CDCl₃, δ=7.26, 500 MHz) 7.44-7.40 (m, 2H), 7.33-7.24 (m, 6H), 7.21-7.16 (m, 2H), 5.13 (d, J=12.5, 1H), 5.08 (d, J=12.5, 1H), 4.02 (s, 1H), 2.65-2.38 (app br s, 8H), 2.25 (s, 3H). RT=2.10 (Cond. III); >98% homogeneity index; LC/MS: Anal. Calcd. for [M+H]⁺. C₂₀H₂₅N₂O₂: 325.19; found 325.20.

A methanol (10 mL) solution of either enantiomer of benzyl 2-(4-methylpiperazin-1-yl)-2-phenylacetate (1.0 g, 3.1 mmol) was added to a suspension of 10% Pd/C (120 mg) in methanol (5.0 mL). The reaction mixture was exposed to a balloon of hydrogen, under a careful monitoring, for <50 minutes. Immediately after the completion of the reaction, the catalyst was filtered through diatomaceous earth (Celite®) and the filtrate was concentrated in vacuo to provide Cap-7, contaminated with phenylacetic acid as a tan foam (867.6 mg; mass is above the theoretical yield). The product was used for the next step without further purification. ¹H NMR (DMSO-d₆, δ=2.5, 500 MHz) δ 7.44-7.37 (m, 2H), 7.37-7.24 (m, 3H), 3.92 (s, 1H), 2.63-2.48 (app. br s, 2H), 2.48-2.32 (m, 6H), 2.19 (s, 3H); RT=0.31 (Cond. II); >90% homogeneity index; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₃H₁₉N₂O₂: 235.14; found 235.15; HRMS: Anal. Calcd. for [M+H]⁺ C₁₃H₁₉N₂O₂: 235.1447; found 235.1440.

The synthesis of Cap-8 and Cap-9 was conducted according to the synthesis of Cap-7 by using appropriate amines for the SN₂ displacement step (i.e., 4-hydroxypiperidine for Cap-8 and (S)-3-fluoropyrrolidine for Cap-9) and modified conditions for the separation of the respective stereoisomeric intermediates, as described below.

The enantiomeric separation of the intermediate benzyl 2-(4-hydroxypiperidin-1-yl)-2-phenyl acetate was effected by employing the following conditions: the compound (500 mg) was dissolved in ethanol/heptane (5 mL/45 mL). The resulting solution was injected (5 mL/injection) on a chiral HPLC column (Chiracel OJ, 2 cm ID×25 cm L, 10 μm) eluting with 80:20 heptane/ethanol at 10 mL/min, monitored at 220 nm, to provide 186.3 mg of enantiomer-1 and 209.1 mg of enantiomer-2 as light-yellow viscous oils. These benzyl ester was hydrogenolysed according to the preparation of Cap-7 to provide Cap-8: ¹H NMR (DMSO-d₆, δ=2.5, 500 MHz) 7.40 (d, J=7, 2H), 7.28-7.20 (m, 3H), 3.78 (s 1H), 3.46 (m, 1H), 2.93 (m, 1H), 2.62 (m, 1H), 2.20 (m, 2H), 1.70 (m, 2H), 1.42 (m, 2H). RT=0.28 (Cond. II); >98% homogeneity index; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₃H₁₈NO₃: 236.13; found 236.07; HRMS: Calcd. for [M+H]⁺ C₁₃H₁₈NO₃: 236.1287; found

The diastereomeric separation of the intermediate benzyl 2-((S)-3-fluoropyrrolidin-1-yl)-2-phenylacetate was effected by employing the following conditions: the ester (220 mg) was separated on a chiral HPLC column (Chiracel OJ-H, 0.46 cm ID×25 cm L, 5 μm) eluting with 95% CO₂/5% methanol with 0.1% TFA, at 10 bar pressure, 70 mL/min flow rate, and a temperature of 35° C. The HPLC elute for the respective stereiosmers was concentrated, and the residue was dissolved in CH₂Cl₂ (20 mL) and washed with an aqueous medium (10 mL water +1 mL saturated NaHCO₃ solution). The organic phase was dried (MgSO₄), filtered, and concentrated in vacuo to provide 92.5 mg of fraction-1 and 59.6 mg of fraction-2. These benzyl esters were hydrogenolysed according to the preparation of Cap-7 to prepare Caps 9a and 9b. Cap-9a (diastereomer-1; the sample is a TFA salt as a result of purification on a reverse phase HPLC using H₂O/methanol/TFA solvent): ¹H NMR (DMSO-d₆, δ=2.5, 400 MHz) 7.55-7.48 (m, 5H), 5.38 (d of m, J=53.7, 1H), 5.09 (br s, 1H), 3.84-2.82 (br m, 4H), 2.31-2.09 (m, 2H). RT=0.42 (Cond. I); >95% homogeneity index; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₂H₁₅FNO₂: 224.11; found 224.14; Cap-9b (diastereomer-2): ¹H NMR (DMSO-d₆, δ=2.5, 400 MHz) 7.43-7.21 (m, 5H), 5.19 (d of m, J=55.9, 1H), 3.97 (s, 1H), 2.95-2.43 (m, 4H), 2.19-1.78 (m, 2H). RT=0.44 (Cond. I); LC/MS: Anal. Calcd. for [M+H]⁺ C₁₂H₁₅FNO₂: 224.11; found 224.14.

To a solution of D-proline (2.0 g, 17 mmol) and formaldehyde (2.0 mL of 37% wt. in H₂O) in methanol (15 mL) was added a suspension of 10% Pd/C (500 mg) in methanol (5 mL). The mixture was stirred under a balloon of hydrogen for 23 hours. The reaction mixture was filtered through diatomaceous earth (Celite®) and concentrated in vacuo to provide Cap-10 as an off-white solid (2.15 g). ¹H NMR (DMSO-d₆, δ=2.5, 500 MHz) 3.42 (m, 1H), 3.37 (dd, J=9.4, 6.1, 1H), 2.85-2.78 (m, 1H), 2.66 (s, 3H), 2.21-2.13 (m, 1H), 1.93-1.84 (m, 2H), 1.75-1.66 (m, 1H). RT=0.28 (Cond. II); >98% homogeneity index; LC/MS: Anal. Calcd. for [M+H]⁺ C₆H₁₂NO₂: 130.09; found 129.96.

A mixture of (2S,4R)-4-fluoropyrrolidine-2-carboxylic acid (0.50 g, 3.8 mmol), formaldehyde (0.5 mL of 37% wt. in H₂O), 12 N HCl (0.25 mL) and 10% Pd/C (50 mg) in methanol (20 mL) was stirred under a balloon of hydrogen for 19 hours. The reaction mixture was filtered through diatomaceous earth (Celite®) and the filtrate was concentrated in vacuo. The residue was recrystallized from isopropanol to provide the HCl salt of Cap-11 as a white solid (337.7 mg). ¹H NMR (DMSO-d₆, δ=2.5, 500 MHz) 5.39 (d m, J=53.7, 1H), 4.30 (m, 1H), 3.90 (ddd, J=31.5, 13.5, 4.5, 1H), 3.33 (dd, J=25.6, 13.4, 1H), 2.85 (s, 3H), 2.60-2.51 (m, 1H), 2.39-2.26 (m, 1H). RT=0.28 (Cond. II); >98% homogeneity index; LC/MS: Anal. Calcd. for [M+H]⁺ C₆H₁₁FNO₂: 148.08; found 148.06.

L-Alanine (2.0 g, 22.5 mmol) was dissolved in 10% aqueous sodium carbonate solution (50 mL), and a THF (50 mL) solution of methyl chloroformate (4.0 mL) was added to it. The reaction mixture was stirred under ambient conditions for 4.5 hours and concentrated in vacuo. The resulting white solid was dissolved in water and acidified with 1N HCl to a pH ˜2-3. The resulting solutions was extracted with ethyl acetate (3×100 mL), and the combined organic phase was dried (Na₂SO₄), filtered, and concentrated in vacuo to provide a colorless oil (2.58 g). 500 mg of this material was purified by a reverse phase HPLC(H₂O/methanol/TFA) to provide 150 mg of Cap-12 as a colorless oil. ¹H NMR (DMSO-d₆, δ=2.5, 500 MHz) 7.44 (d, J=7.3, 0.8H), 7.10 (br s, 0.2H), 3.97 (m, 1H), 3.53 (s, 3H), 1.25 (d, J=7.3, 3H).

A mixture of L-alanine (2.5 g, 28 mmol), formaldehyde (8.4 g, 37 wt. %), 1N HCl (30 mL) and 10% Pd/C (500 mg) in methanol (30 mL) was stirred under a hydrogen atmosphere (50 psi) for 5 hours. The reaction mixture was filtered through diatomaceous earth (Celite®) and the filtrate was concentrated in vacuo to provide the HCl salt of Cap-13 as an oil which solidified upon standing under vacuum (4.4 g; the mass is above theoretical yield). The product was used without further purification. ¹H NMR (DMSO-d₆, δ=2.5, 500 MHz) δ 12.1 (br s, 1H), 4.06 (q, J=7.4, 1H), 2.76 (s, 6H), 1.46 (d, J=7.3, 3H).

Step 1: A mixture of (R)-(−)-D-phenylglycine tert-butyl ester (3.00 g, 12.3 mmol), NaBH₃CN (0.773 g, 12.3 mmol), KOH (0.690 g, 12.3 mmol) and acetic acid (0.352 mL, 6.15 mmol) were stirred in methanol at 0° C. To this mixture was added glutaric dialdehyde (2.23 mL, 12.3 mmol) dropwise over 5 minutes. The reaction mixture was stirred as it was allowed to warm to ambient temperature and stirring was continued at the same temperature for 16 hours. The solvent was subsequently removed and the residue was partitioned with 10% aqueous NaOH and ethyl acetate. The organic phase was separated, dried (MgSO₄), filtered and concentrated to dryness to provide a clear oil. This material was purified by reverse-phase preparative HPLC (Primesphere C-18, 30×100 mm; CH₃CN—H₂O—0.1% TFA) to give the intermediate ester (2.70 g, 56%) as a clear oil. ¹H NMR (400 MHz, CDCl₃) δ 7.53-7.44 (m, 3H), 7.40-7.37 (m, 2H), 3.87 (d, J=10.9 Hz, 1H), 3.59 (d, J=10.9 Hz, 1H), 2.99 (t, J=11.2 Hz, 1H), 2.59 (t, J=11.4 Hz, 1H), 2.07-2.02 (m, 2H), 1.82 (d, J=1.82 Hz, 3H), 1.40 (s, 9H). LC/MS: Anal. Calcd. for C₁₇H₂₅NO₂: 275; found: 276 (M+H)⁺.

Step 2: To a stirred solution of the intermediate ester (1.12 g, 2.88 mmol) in dichloromethane (10 mL) was added TFA (3 mL). The reaction mixture was stirred at ambient temperature for 4 hours and then it was concentrated to dryness to give a light yellow oil. The oil was purified using reverse-phase preparative HPLC (Primesphere C-18, 30×100 mm; CH₃CN—H₂O—0.1% TFA). The appropriate fractions were combined and concentrated to dryness in vacuo. The residue was then dissolved in a minimum amount of methanol and applied to applied to MCX LP extraction cartridges (2×6 g). The cartridges were rinsed with methanol (40 mL) and then the desired compound was eluted using 2M ammonia in methanol (50 mL). Product-containing fractions were combined and concentrated and the residue was taken up in water. Lyophilization of this solution provided the title compound (0.492 g, 78%) as a light yellow solid. ¹H NMR (DMSO-d₆) δ 7.50 (s, 5H), 5.13 (s, 1H), 3.09 (br s, 2H), 2.92-2.89 (m, 2H), 1.74 (m, 4H), 1.48 (br s, 2H). LC/MS: Anal. Calcd. for C₁₃H₁₇NO₂: 219; found: 220 (M+H)⁺.

Step 1: (S)-1-Phenylethyl 2-bromo-2-phenylacetate: To a mixture of α-bromophenylacetic acid (10.75 g, 0.050 mol), (S)-(−)-1-phenylethanol (7.94 g, 0.065 mol) and DMAP (0.61 g, 5.0 mmol) in dry dichloromethane (100 mL) was added solid EDCI (12.46 g, 0.065 mol) all at once. The resulting solution was stirred at room temperature under Ar for 18 hours and then it was diluted with ethyl acetate, washed (H₂O×2, brine), dried (Na₂SO₄), filtered, and concentrated to give a pale yellow oil. Flash chromatography (SiO₂/hexane-ethyl acetate, 4:1) of this oil provided the title compound (11.64 g, 73%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.53-7.17 (m, 10H), 5.95 (q, J=6.6 Hz, 0.5H), 5.94 (q, J=6.6 Hz, 0.5H), 5.41 (s, 0.5H), 5.39 (s, 0.5H), 1.58 (d, J=6.6 Hz, 1.5H), 1.51 (d, J=6.6 Hz, 1.5H).

Step 2: (S)-1-Phenylethyl (R)-2-(4-hydroxy-4-methylpiperidin-1-yl)-2-phenylacetate: To a solution of (S)-1-phenylethyl 2-bromo-2-phenylacetate (0.464 g, 1.45 mmol) in THF (8 mL) was added triethylamine (0.61 mL, 4.35 mmol), followed by tetrabutylammonium iodide (0.215 g, 0.58 mmol). The reaction mixture was stirred at room temperature for 5 minutes and then a solution of 4-methyl-4-hydroxypiperidine (0.251 g, 2.18 mmol) in THF (2 mL) was added. The mixture was stirred for 1 hour at room temperature and then it was heated at 55-60° C. (oil bath temperature) for 4 hours. The cooled reaction mixture was then diluted with ethyl acetate (30 mL), washed (H₂O×2, brine), dried (MgSO₄), filtered and concentrated. The residue was purified by silica gel chromatography (0-60% ethyl acetate-hexane) to provide first the (S,R)-isomer of the title compound (0.306 g, 60%) as a white solid and then the corresponding (S,S)-isomer (0.120 g, 23%), also as a white solid. (S,R)-isomer: ¹H NMR (CD₃OD) δ 7.51-7.45 (m, 2H), 7.41-7.25 (m, 8H), 5.85 (q, J=6.6 Hz, 1H), 4.05 (s, 1H), 2.56-2.45 (m, 2H), 2.41-2.29 (m, 2H), 1.71-1.49 (m, 4H), 1.38 (d, J=6.6 Hz, 3H), 1.18 (s, 3H). LCMS: Anal. Calcd. for C₂₂H₂₇NO₃: 353; found: 354 (M+H)⁺. (S,S)-isomer: ¹H NMR (CD₃OD) δ 7.41-7.30 (m, 5H), 7.20-7.14 (m, 3H), 7.06-7.00 (m, 2H), 5.85 (q, J=6.6 Hz, 1H), 4.06 (s, 1H), 2.70-2.60 (m, 1H), 2.51 (dt, J=6.6, 3.3 Hz, 1H), 2.44-2.31 (m, 2H), 1.75-1.65 (m, 1H), 1.65-1.54 (m, 3H), 1.50 (d, J=6.8 Hz, 3H), 1.20 (s, 3H). LCMS: Anal. Calcd. for C₂₂H₂₇NO₃: 353; found: 354 (M+H)⁺.

Step 3: (R)-2-(4-Hydroxy-4-methylpiperidin-1-yl)-2-phenylacetic acid: To a solution of (S)-1-phenylethyl (R)-2-(4-hydroxy-4-methylpiperidin-1-yl)-2-phenylacetate (0.185 g, 0.52 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (1 mL) and the mixture was stirred at room temperature for 2 hours. The volatiles were subsequently removed in vacuo and the residue was purified by reverse-phase preparative HPLC (Primesphere C-18, 20×100 mm; CH₃CN—H₂O—0.1% TFA) to give the title compound (as TFA salt) as a pale bluish solid (0.128 g, 98%). LCMS: Anal. Calcd. for C₁₄H₁₉NO₃: 249; found: 250 (M+H)⁺.

Step 1: (S)-1-Phenylethyl 2-(2-fluorophenyl)acetate: A mixture of 2-fluorophenylacetic acid (5.45 g, 35.4 mmol), (S)-1-phenylethanol (5.62 g, 46.0 mmol), EDCI (8.82 g, 46.0 mmol) and DMAP (0.561 g, 4.60 mmol) in CH₂Cl₂ (100 mL) was stirred at room temperature for 12 hours. The solvent was then concentrated and the residue partitioned with H₂O-ethyl acetate. The phases were separated and the aqueous layer back-extracted with ethyl acetate (2×). The combined organic phases were washed (H₂O, brine), dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (Biotage/0-20% ethyl acetate-hexane) to provide the title compound as a colorless oil (8.38 g, 92%). ¹H NMR (400 MHz, CD₃OD) δ 7.32-7.23 (m, 7H), 7.10-7.04 (m, 2), 5.85 (q, J=6.5 Hz, 1H), 3.71 (s, 2H), 1.48 (d, J=6.5 Hz, 3H).

Step 2: (R)—((S)-1-Phenylethyl) 2-(2-fluorophenyl)-2-(piperidin-1-yl)acetate: To a solution of (S)-1-phenylethyl 2-(2-fluorophenyl)acetate (5.00 g, 19.4 mmol) in THF (1200 mL) at 0° C. was added DBU (6.19 g, 40.7 mmol) and the solution was allowed to warm to room temperature while stirring for 30 minutes. The solution was then cooled to −78° C. and a solution of CBr₄ (13.5 g, 40.7 mmol) in THF (100 mL) was added and the mixture was allowed to warm to −10° C. and stirred at this temperature for 2 hours. The reaction mixture was quenched with saturated aq. NH₄Cl and the layers were separated. The aqueous layer was back-extracted with ethyl acetate (2×) and the combined organic phases were washed (H₂O, brine), dried (Na₂SO₄), filtered, and concentrated in vacuo. To the residue was added piperidine (5.73 mL, 58.1 mmol) and the solution was stirred at room temperature for 24 hours. The volatiles were then concentrated in vacuo and the residue was purified by silica gel chromatography (Biotage/0-30% diethyl ether-hexane) to provide a pure mixture of diastereomers (2:1 ratio by ¹H NMR) as a yellow oil (2.07 g, 31%), along with unreacted starting material (2.53 g, 51%). Further chromatography of the diastereomeric mixture (Biotage/0-10% diethyl ether-toluene) provided the title compound as a colorless oil (0.737 g, 11%). ¹H NMR (400 MHz, CD₃OD) δ 7.52 (ddd, J=9.4, 7.6, 1.8 Hz, 1H), 7.33-7.40 (m, 1), 7.23-7.23 (m, 4H), 7.02-7.23 (m, 4H), 5.86 (q, J=6.6 Hz, 1H), 4.45 (s, 1H), 2.39-2.45 (m, 4H), 1.52-1.58 (m, 4H), 1.40-1.42 (m, 1H), 1.38 (d, J=6.6 Hz, 3H). LCMS: Anal. Calcd. for C₂₁H₂₄FNO₂: 341; found: 342 (M+H)⁺.

Step 3: (R)-2-(2-fluorophenyl)-2-(piperidin-1-yl)acetic acid: A mixture of (R)—((S)-1-phenylethyl) 2-(2-fluorophenyl)-2-(piperidin-1-yl)acetate (0.737 g, 2.16 mmol) and 20% Pd(OH)₂/C (0.070 g) in ethanol (30 mL) was hydrogenated at room temperature and atmospheric pressure (H₂ balloon) for 2 hours. The solution was then purged with Ar, filtered through diatomaceous earth (Celite®), and concentrated in vacuo. This provided the title compound as a colorless solid (0.503 g, 98%). ¹H NMR (400 MHz, CD₃OD) δ 7.65 (ddd, J=9.1, 7.6, 1.5 Hz, 1H), 7.47-7.53 (m, 1H), 7.21-7.30 (m, 2H), 3.07-3.13 (m, 4H), 1.84 (br s, 4H), 1.62 (br s, 2H). LCMS: Anal. Calcd. for C₁₃H₁₆FNO₂: 237; found: 238 (M+H)⁺.

Step 1: (S)-1-Phenylethyl (R)-2-(4-hydroxy-4-phenylpiperidin-1-yl)-2-phenylacetate: To a solution of (S)-1-phenylethyl 2-bromo-2-phenylacetate (1.50 g, 4.70 mmol) in THF (25 mL) was added triethylamine (1.31 mL, 9.42 mmol), followed by tetrabutylammonium iodide (0.347 g, 0.94 mmol). The reaction mixture was stirred at room temperature for 5 minutes and then a solution of 4-phenyl-4-hydroxypiperidine (1.00 g, 5.64 mmol) in THF (5 mL) was added. The mixture was stirred for 16 hours and then it was diluted with ethyl acetate (100 mL), washed (H₂O×2, brine), dried (MgSO₄), filtered and concentrated. The residue was purified on a silica gel column (0-60% ethyl acetate-hexane) to provide an approximately 2:1 mixture of diastereomers, as judged by ¹H NMR. Separation of these isomers was performed using supercritical fluid chromatography (Chiralcel OJ-H, 30×250 mm; 20% ethanol in CO₂ at 35° C.), to give first the (R)-isomer of the title compound (0.534 g, 27%) as a yellow oil and then the corresponding (S)-isomer (0.271 g, 14%), also as a yellow oil. (S,R)-isomer: ¹H NMR (400 MHz, CD₃OD) δ 7.55-7.47 (m, 4H), 7.44-7.25 (m, 10H), 7.25-7.17 (m, 1H), 5.88 (q, J=6.6 Hz, 1H), 4.12 (s, 1H), 2.82-2.72 (m, 1H), 2.64 (dt, J=11.1, 2.5 Hz, 1H), 2.58-2.52 (m, 1H), 2.40 (dt, J=11.1, 2.5 Hz, 1H), 2.20 (dt, J=12.1, 4.6 Hz, 1H), 2.10 (dt, J=12.1, 4.6 Hz, 1H), 1.72-1.57 (m, 2H), 1.53 (d, J=6.5 Hz, 3H). LCMS: Anal. Calcd. for C₂₇H₂₉NO₃: 415; found: 416 (M+H)⁺; (S,S)-isomer: H¹NMR (400 MHz, CD₃OD) δ 7.55-7.48 (m, 2H), 7.45-7.39 (m, 2H), 7.38-7.30 (m, 5H), 7.25-7.13 (m, 4H), 7.08-7.00 (m, 2H), 5.88 (q, J=6.6 Hz, 1H), 4.12 (s, 1H), 2.95-2.85 (m, 1H), 2.68 (dt, J=11.1, 2.5 Hz, 1H), 2.57-2.52 (m, 1H), 2.42 (dt, J=11.1, 2.5 Hz, 1H), 2.25 (dt, J=12.1, 4.6 Hz, 1H), 2.12 (dt, J=12.1, 4.6 Hz, 1H), 1.73 (dd, J=13.6, 3.0 Hz, 1H), 1.64 (dd, J=13.6, 3.0 Hz, 1H), 1.40 (d, J=6.6 Hz, 3H). LCMS: Anal. Calcd. for C₂₇H₂₉NO₃: 415; found: 416 (M+H)⁺.

The following esters were prepared in similar fashion:

Compound Structure Analytical Data Intermediate- 17b

Diasteromer 1: RT = 11.76 minutes (Cond'n II); LCMS: Anal. Calcd. for: C₂₀H₂₂N₂O₃ 338.16 Found: 339.39 (M + H)⁺; Diastereomer 2: RT = 10.05 minutes (Cond'n II); LCMS: Anal. Calcd. for: C₂₀H₂₂N₂O₃ 338.16; Found: 339.39 (M + H)⁺. Intermediate- 17c

Diastereomer 1: T_(R) = 4.55 minutes (Cond'n I); LCMS: Anal. Calcd. for: C₂₁H₂₆N₂O₂ 338.20 Found: 339.45 (M + H)⁺; Diastereomer 2: T_(R) = 6.00 minutes (Cond'n I); LCMS: Anal. Calcd. for: C₂₁H₂₆N₂O₂ 338.20 Found: 339.45 (M + H)⁺. Intermediate- 17d

Diastereomer 1: RT = 7.19 minutes (Cond'n I); LCMS: Anal. Calcd. for: C₂₇H₂₉NO₂ 399.22 Found: 400.48 (M + H)⁺; Diastereomer 2: RT = 9.76 minutes (Cond'n I); LCMS: Anal. Calcd. for: C₂₇H₂₉NO₂ 399.22 Found: 400.48 (M + H)⁺.

Chiral SFC Conditions for determining retention time

Condition I

-   Column: Chiralpak AD-H Column, 4.62×50 mm, 5 μm -   Solvents: 90% CO2-10% methanol with 0.1% DEA -   Temp: 35° C. -   Pressure: 150 bar -   Flow rate: 2.0 mL/min. -   UV monitored @ 220 nm -   Injection: 1.0 mg/3 mL methanol

Condition II

-   Column: Chiralcel OD-H Column, 4.62×50 mm, 5 μm -   Solvents: 90% CO2-10% methanol with 0.1% DEA -   Temp: 35° C. -   Pressure: 150 bar -   Flow rate: 2.0 mL/min. -   UV monitored @ 220 nm -   Injection: 1.0 mg/mL methanol

Cap 17, Step 2; (R)-2-(4-Hydroxy-4-phenylpiperidin-1-yl)-2-phenylacetic acid: To a solution of (5)-1-phenylethyl (R)-2-(4-hydroxy-4-phenylpiperidin-1-yl)-2-phenylacetate (0.350 g, 0.84 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (1 mL) and the mixture was stirred at room temperature for 2 hours. The volatiles were subsequently removed in vacuo and the residue was purified by reverse-phase preparative HPLC (Primesphere C-18, 20×100 mm; CH₃CN—H₂O—0.1% TFA) to give the title compound (as TFA salt) as a white solid (0.230 g, 88%). LCMS: Anal. Calcd. for C₁₉H₂₁NO₃: 311.15; found: 312 (M+H)⁺.

The following carboxylic acids were prepared in optically pure form in a similar fashion:

Cap Structure Analytical Data Cap-17b

RT = 0.27 (Cond'n III); LCMS: Anal. Calcd. for C₁₂H₁₄N₂O₃ 234.10; Found: 235.22 (M + H)⁺. Cap-17c

RT = 0.48 (Cond'n II); LCMS: Anal. Calcd. for C₁₃H₁₈N₂O₂ 234.14; Found: 235.31 (M + H)⁺. Cap-17d

RT = 2.21 (Cond'n I); LCMS: Anal. Calcd. for C₁₉H₂₁NO₂ 295.16; Found: 296.33 (M + H)⁺.

LCMS Conditions for determining retention time

Condition I

-   Column: Phenomenex-Luna 4.6×50 mm S10 -   Start % B=0 -   Fianl % B=100 -   Gradient Time=4 min -   Flow Rate=4 mL/min -   Wavelength=220 -   Solvent A=10% methanol—90% H₂O—0.1% TFA -   Solvent B=90% methanol—10% H₂O—0.1% TFA

Condition II

-   Column: Waters-Sunfire 4.6×50 mm S5 -   Start % 13=0 -   Fianl % B=100 -   Gradient Time=2 min -   Flow Rate=4 mL/min -   Wavelength=220 -   Solvent A=10% methanol—90% H₂O—0.1% TFA -   Solvent B=90% methanol—10% H₂O—0.1% TFA

Condition III

-   Column: Phenomenex 10μ 3.0×50 mm -   Start % B=0 -   Fianl % B=100 -   Gradient Time=2 min -   Flow Rate=4 mL/min -   Wavelength=220 -   Solvent A=10% methanol—90% H₂O—0.1% TFA -   Solvent B=90% methanol—10% H₂O—0.1% TFA

Step 1; (R,S)-Ethyl 2-(4-pyridyl)-2-bromoacetate: To a solution of ethyl 4-pyridylacetate (1.00 g, 6.05 mmol) in dry THF (150 mL) at 0° C. under argon was added DBU (0.99 mL, 6.66 mmol). The reaction mixture was allowed to warm to room temperature over 30 minutes and then it was cooled to −78° C. To this mixture was added CBr₄ (2.21 g, 6.66 mmol) and stirring was continued at −78° C. for 2 hours. The reaction mixture was then quenched with sat. aq. NH₄Cl and the phases were separated. The organic phase was washed (brine), dried (Na₂SO₄), filtered, and concentrated in vacuo. The resulting yellow oil was immediately purified by flash chromatography (SiO₂/hexane-ethyl acetate, 1:1) to provide the title compound (1.40 g, 95%) as a somewhat unstable yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.62 (dd, J=4.6, 1.8 Hz, 2H), 7.45 (dd, J=4.6, 1.8 Hz, 2H), 5.24 (s, 1H), 4.21-4.29 (m, 2H), 1.28 (t, J=7.1 Hz, 3H). LCMS: Anal. Calcd. for C₉H₁₀BrNO₂: 242, 244; found: 243, 245 (M+H)⁺.

Step 2; (R,S)-Ethyl 2-(4-pyridyl)-2-(N,N-dimethylamino)acetate: To a solution of (R,S)-ethyl 2-(4-pyridyl)-2-bromoacetate (1.40 g, 8.48 mmol) in DMF (10 mL) at room temperature was added dimethylamine (2M in THF, 8.5 mL, 17.0 mmol). After completion of the reaction (as judged by thin layer chromatography) the volatiles were removed in vacuo and the residue was purified by flash chromatography (Biotage, 40+M SiO₂ column; 50%-100% ethyl acetate-hexane) to provide the title compound (0.539 g, 31%) as a light yellow oil. NMR (400 MHz, CDCl₃) δ 8.58 (d, J=6.0 Hz, 2H), 7.36 (d, J=6.0 Hz, 2H), 4.17 (m, 2H), 3.92 (s, 1H), 2.27 (s, 6H), 1.22 (t, J=7.0 Hz). LCMS: Anal. Calcd. for C₁₁H₁₆N₂O₂: 208; found: 209 (M+H)⁺.

Step 3; (R,S)-2-(4-Pyridyl)-2-(N,N-dimethylamino)acetic acid: To a solution of (R,S)-ethyl 2-(4-pyridyl)-2-(N,N-dimethylamino)acetate (0.200 g, 0.960 mmol) in a mixture of THF-methanol-H₂O (1:1:1, 6 mL) was added powdered LiOH (0.120 g, 4.99 mmol) at room temperature. The solution was stirred for 3 hours and then it was acidified to pH 6 using 1N HCl. The aqueous phase was washed with ethyl acetate and then it was lyophilized to give the dihydrochloride of the title compound as a yellow solid (containing LiCl). The product was used as such in subsequent steps. ¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (d, J=5.7 Hz, 2H), 7.34 (d, J=5.7 Hz, 2H), 3.56 (s, 1H), 2.21 (s, 6H).

The following examples were prepared in similar fashion using the method described above:

Cap Structure Analytical Data Cap-20

LCMS: no ionization. ¹H NMR (400 MHz, CD₃OD) δ 8.55 (d, J = 4.3 Hz, 1H), 7.84 (app t, J = 5.3 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.37 (app t, J = 5.3 Hz, 1H), 4.35 (s, 1H), 2.60 (s, 6H). Cap-21

LCMS: Anal. Calcd. for C₉H₁₁N₂O₂: 214, 216; found: 215, 217 (M + H)⁺. Cap-22

LCMS: Anal. Calcd. for C₁₀H₁₂N₂O₄: 224; found: 225 (M + H)⁺. Cap-23

LCMS: Anal. Calcd. for C₁₄H₁₅NO₂: 229; found: 230 (M + H)⁺. Cap-24

LCMS: Anal. Calcd. for C₁₁H₁₂F₃NO₂: 247; found: 248 (M + H)⁺. Cap-25

LCMS: Anal. Calcd. for C₁₁H₁₂F₃NO₂: 247; found: 248 (M + H)⁺. Cap-26

LCMS: Anal. Calcd. for C₁₀H₁₂FNO₂: 197; found: 198 (M + H)⁺. Cap-27

LCMS: Anal. Calcd. for C₁₀H₁₂FNO₂: 247; found: 248 (M + H)⁺. Cap-28

LCMS: Anal. Calcd. for C₁₀H₁₂ClNO₂: 213; found: 214 (M + H)⁺. Cap-29

LCMS: Anal. Calcd. for C₁₀H₁₂ClNO₂: 213; found: 214 (M + H)⁺. Cap-30

LCMS: Anal. Calcd. for C₁₀H₁₂ClNO₂: 213; found: 214 (M + H)⁺. Cap-31

LCMS: Anal. Calcd. for C₈H₁₂N₂O₂S: 200; found: 201 (M + H)⁺. Cap-32

LCMS: Anal. Calcd. for C₈H₁₁NO₂S: 185; found: 186 (M + H)⁺. Cap-33

LCMS: Anal. Calcd. for C₈H₁₁NO₂S: 185; found: 186 (M + H)⁺. Cap-34

LCMS: Anal. Calcd. for C₁₁H₁₂N₂O₃: 220; found: 221 (M + H)⁺. Cap-35

LCMS: Anal. Calcd. for C₁₂H₁₃NO₂S: 235; found: 236 (M + H)⁺. Cap-36

LCMS: Anal. Calcd. for C₁₂H₁₄N₂O₂S: 250; found: 251 (M + H)⁺.

Step 1; (R,S)-Ethyl 2-(quinolin-3-yl)-2-(N,N-dimethylamino)-acetate: A mixture of ethyl N,N-dimethylaminoacetate (0.462 g, 3.54 mmol), K₃PO₄ (1.90 g, 8.95 mmol), Pd(t-Bu₃P)₂ (0.090 g, 0.176 mmol) and toluene (10 mL) was degassed with a stream of Ar bubbles for 15 minutes. The reaction mixture was then heated at 100° C. for 12 hours, after which it was cooled to room temperature and poured into H₂O. The mixture was extracted with ethyl acetate (2×) and the combined organic phases were washed (H₂O, brine), dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified first by reverse-phase preparative HPLC (Primesphere C-18, 30×100 mm; CH₃CN—H₂O—5 mM NH₄OAc) and then by flash chromatography (SiO₂/hexane-ethyl acetate, 1:1) to provide the title compound (0.128 g, 17%) as an orange oil. NMR (400 MHz, CDCl₃) δ 8.90 (d, J=2.0 Hz, 1H), 8.32 (d, J=2.0 Hz, 1H), 8.03-8.01 (m, 2H), 7.77 (ddd, J=8.3, 6.8, 1.5 Hz, 1H), 7.62 (ddd, J=8.3, 6.8, 1.5 Hz, 1H), 4.35 (s, 1H), 4.13 (m, 2H), 2.22 (s, 6H), 1.15 (t, J=7.0 Hz, 3H). LCMS: Anal. Calcd. for C₁₅H₁₈N₂O₂: 258; found: 259 (M+H)⁺.

Step 2; (R,S) 2-(Quinolin-3-yl)-2-(N,N-dimethylamino)-acetic acid: A mixture of (R,S)-ethyl 2-(quinolin-3-yl)-2-(N,N-dimethylamino)acetate (0.122 g, 0.472 mmol) and 6M HCl (3 mL) was heated at 100° C. for 12 hours. The solvent was removed in vacuo to provide the dihydrochloride of the title compound (0.169 g, >100%) as a light yellow foam. The unpurified material was used in subsequent steps without further purification. LCMS: Anal. Calcd. for C₁₃H₁₄N₂O₂: 230; found: 231 (M+H)⁺.

Step 1; (R)—((S)-1-phenylethyl) 2-(dimethylamino)-2-(2-fluorophenyl)acetate and (S)—((S)-1-phenylethyl) 2-(dimethylamino)-2-(2-fluorophenyl)acetate: To a mixture of (RS)-2-(dimethylamino)-2-(2-fluorophenyl)acetic acid (2.60 g, 13.19 mmol), DMAP (0.209 g, 1.71 mmol) and (S)-1-phenylethanol (2.09 g, 17.15 mmol) in CH₂Cl₂ (40 mL) was added EDCI (3.29 g, 17.15 mmol) and the mixture was allowed to stir at room temperature for 12 hours. The solvent was then removed in vacuo and the residue partitioned with ethyl acetate-H₂O. The layers were separated, the aqueous layer was back-extracted with ethyl acetate (2×) and the combined organic phases were washed (H₂O, brine), dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (Biotage/0-50% diethyl ether-hexane). The resulting pure diastereomeric mixture was then separated by reverse-phase preparative HPLC (Primesphere C-18, 30×100 mm; CH₃CN—H₂O—0.1% TFA) to give first (S)-1-phenethyl (R)-2-(dimethylamino)-2-(2-fluorophenyl)acetate (0.501 g, 13%) and then (S)-1-phenethyl (S)-2-(dimethylamino)-2-(2-fluorophenyl)-acetate (0.727 g. 18%), both as their TFA salts. (S,R)-isomer: ¹H NMR (400 MHz, CD₃OD) δ 7.65-7.70 (m, 1H), 7.55-7.60 (ddd, J=9.4, 8.1, 1.5 Hz, 1H), 7.36-7.41 (m, 2H), 7.28-7.34 (m, 5H), 6.04 (q, J=6.5 Hz, 1H), 5.60 (s, 1H), 2.84 (s, 6H), 1.43 (d, J=6.5 Hz, 3H). LCMS: Anal. Calcd. for C₁₈H₂₀FNO₂: 301; found: 302 (M+H)⁴; (S,S)-isomer: ¹H NMR (400 MHz, CD₃OD) δ 7.58-7.63 (m, 1H), 7.18-7.31 (m, 6H), 7.00 (dd, J=8.5, 1.5 Hz, 2H), 6.02 (q, J=6.5 Hz, 1H), 5.60 (s, 1H), 2.88 (s, 6H), 1.54 (d, J=6.5 Hz, 3H). LCMS: Anal. Calcd. for C₁₈H₂₀FNO₂: 301; found: 302 (M+H)⁺.

Step 2; (R)-2-(dimethylamino)-2-(2-fluorophenyl)acetic acid: A mixture of (R)—((S)-1-phenylethyl) 2-(dimethylamino)-2-(2-fluorophenyl)acetate TFA salt (1.25 g, 3.01 mmol) and 20% Pd(OH)₂/C (0.125 g) in ethanol (30 mL) was hydrogenated at room temperature and atmospheric pressure (H₂ balloon) for 4 hours. The solution was then purged with Ar, filtered through diatomaceous earth (Celite®), and concentrated in vacuo. This gave the title compound as a colorless solid (0.503 g, 98%). ¹H NMR (400 MHz, CD₃OD) δ 7.53-7.63 (m, 2H), 7.33-7.38 (m, 2H), 5.36 (s, 1H), 2.86 (s, 6H). LCMS: Anal. Calcd. for C₁₀H₁₂FNO₂: 197; found: 198 (M+H)⁺.

The S-isomer could be obtained from (S)—((S)-1-phenylethyl) 2-(dimethylamino)-2-(2-fluorophenyl)acetate TFA salt in similar fashion.

A mixture of (R)-(2-chlorophenyl)glycine (0.300 g, 1.62 mmol), formaldehyde (35% aqueous solution, 0.80 mL, 3.23 mmol) and 20% Pd(OH)₂/C (0.050 g) was hydrogenated at room temperature and atmospheric pressure (H₂ balloon) for 4 hours. The solution was then purged with Ar, filtered through diatomaceous earth (Celite®) and concentrated in vacuo. The residue was purified by reverse-phase preparative HPLC (Primesphere C-18, 30×100 mm; CH₃CN—H₂O—0.1% TFA) to give the TFA salt of the title compound (R)-2-(dimethylamino)-2-(2-chlorophenyl)acetic acid as a colorless oil (0.290 g, 55%). ¹H NMR (400 MHz, CD₃OD) δ 7.59-7.65 (m, 2H), 7.45-7.53 (m, 2H), 5.40 (s, 1H), 2.87 (s; 6H). LCMS: Anal. Calcd. for C₁₀H₁₂ClNO₂: 213; found: 214 (M+H)⁺.

To an ice-cold solution of (R)-(2-chlorophenyl)glycine (1.00 g, 5.38 mmol) and NaOH (0.862 g, 21.6 mmol) in H₂O (5.5 mL) was added methyl chloroformate (1.00 mL, 13.5 mmol) dropwise. The mixture was allowed to stir at 0° C. for 1 hour and then it was acidified by the addition of conc. HCl (2.5 mL). The mixture was extracted with ethyl acetate (2×) and the combined organic phase was washed (H₂O, brine), dried (Na₂SO₄), filtered, and concentrated in vacuo to give the title compound (R)-2-(methoxycarbonylamino)-2-(2-chlorophenyl)acetic acid as a yellow-orange foam (1.31 g, 96%). ¹H NMR (400 MHz, CD₃OD) δ 7.39-7.43 (m, 2H), 7.29-7.31 (m, 2H), 5.69 (s, 1H), 3.65 (s, 3H). LCMS: Anal. Calcd. for C₁₀H₁₀ClNO₄: 243; found: 244 (M+H)⁺.

To a suspension of 2-(2-(chloromethyl)phenyl)acetic acid (2.00 g, 10.8 mmol) in THF (20 mL) was added morpholine (1.89 g, 21.7 mmol) and the solution was stirred at room temperature for 3 hours. The reaction mixture was then diluted with ethyl acetate and extracted with H₂O (2×). The aqueous phase was lyophilized and the residue was purified by silica gel chromatography (Biotage/0-10% methanol-CH₂Cl₂) to give the title compound 2-(2-(Morpholinomethyl)phenyl)acetic acid as a colorless solid (2.22 g, 87%). ¹H NMR (400 MHz, CD₃OD) δ 7.37-7.44 (m, 3H), 7.29-7.33 (m, 1H), 4.24 (s, 2H), 3.83 (br s, 4H), 3.68 (s, 2H), 3.14 (br s, 4H). LCMS: Anal. Calcd. for C₁₃H₁₇NO₃: 235; found: 236 (M+H)⁺.

The following examples were similarly prepared using the method described for Cap-41:

Cap Structure Analytical Data Cap-43

LCMS: Anal. Calcd. for C₁₃H₁₇NO₂: 219; found: 220 (M + H)⁺. Cap-44

LCMS: Anal. Calcd. for C₁₁H₁₅NO₂: 193; found: 194 (M + H)⁺. Cap-45

LCMS: Anal. Calcd. for C₁₄H₂₀N₂O₂: 248; found: 249 (M + H)⁺.

HMDS (1.85 mL, 8.77 mmol) was added to a suspension of (R)-2-amino-2-phenylacetic acid p-toluenesulfonate (2.83 g, 8.77 mmol) in CH₂Cl₂ (10 mL) and the mixture was stirred at room temperature for 30 minutes. Methyl isocyanate (0.5 g, 8.77 mmol) was added in one portion stirring continued for 30 minutes. The reaction was quenched by addition of H₂O (5 mL) and the resulting precipitate was filtered, washed with H₂O and n-hexanes, and dried under vacuum. (R)-2-(3-methylureido)-2-phenylacetic acid (1.5 g; 82%) was recovered as a white solid and it was used without further purification. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.54 (d, J=4.88 Hz, 3H) 5.17 (d, J=7.93 Hz, 1H) 5.95 (q, J=4.48 Hz, 1H) 6.66 (d, J=7.93 Hz, 1H) 7.26-7.38 (m, 5H) 12.67 (s, 1H). LCMS: Anal. Calcd. for C₁₀H₁₂N₂O₃ 208.08 found 209.121 (M+H)⁺; HPLC Phenomenex C-18 3.0×46 mm, 0 to 100% B over 2 minutes, 1 minute hold time, A=90% water, 10% methanol, 0.1% TFA, B=10% water, 90% methanol, 0.1% TFA, RT=1.38 min, 90% homogeneity index.

The desired product was prepared according to the method described for Cap-45a. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.96 (t, J=7.17 Hz, 3H) 2.94-3.05 (m, 2H) 5.17 (d, J=7.93 Hz, 1H) 6.05 (t, J=5.19 Hz, 1H) 6.60 (d, J=7.63 Hz, 1H) 7.26-7.38 (m, 5H) 12.68 (s, 1H). LCMS: Anal. Calcd. for C₁₁H₁₄N₂O₃ 222.10 found 223.15 (M+H)⁺. HPLC XTERRA C-18 3.0×506 mm, 0 to 100% B over 2 minutes, 1 minute hold time, A=90% water, 10% methanol, 0.2% H₃PO₄, B=10% water, 90% methanol, 0.2% H₃PO₄, RT=0.87 min, 90% homogeneity index.

Step 1; (R)-tert-butyl 2-(3,3-dimethylureido)-2-phenylacetate: To a stirred solution of (R)-tert-butyl-2-amino-2-phenylacetate (1.0 g, 4.10 mmol) and Hunig's base (1.79 mL, 10.25 mmol) in DMF (40 mL) was added dimethylcarbamoyl chloride (0.38 mL, 4.18 mmol) dropwise over 10 minutes. After stirring at room temperature for 3 hours, the reaction was concentrated under reduced pressure and the resulting residue was dissolved in ethyl acetate. The organic layer was washed with H₂O, 1N aq. HCl and brine, dried (MgSO₄), filtered and concentrated under reduced pressure. (R)-tert-butyl 2-(3,3-dimethylureido)-2-phenylacetate was obtained as a white solid (0.86 g; 75%) and used without further purification. ¹H NMR (500 MHz, DMSO-d₅) δ ppm 1.33 (s, 9H) 2.82 (s, 6H) 5.17 (d, J=7.63 Hz, 1H) 6.55 (d, J=7.32 Hz, 1H) 7.24-7.41 (m, 5H). LCMS: Anal. Calcd. for C₁₅H₂₂N₂O₃ 278.16 found 279.23 (M+H)⁺; HPLC Phenomenex LUNA C-18 4.6×50 mm, 0 to 100% B over 4 minutes, 1 minute hold time, A=90% water, 10% methanol, 0.1% TFA, B=10% water, 90% methanol, 0.1% TFA, RT=2.26 min, 97% homogeneity index.

Step 2; (R)-2-(3,3-dimethylureido)-2-phenylacetic acid: To a stirred solution of ((R)-tert-butyl 2-(3,3-dimethylureido)-2-phenylacetate (0.86 g, 3.10 mmol) in CH₂Cl₂ (250 mL) was added TFA (15 mL) dropwise and the resulting solution was stirred at rt for 3 hours. The desired compound was then precipitated out of solution with a mixture of EtOAC:Hexanes (5:20), filtered off and dried under reduced pressure. (R)-2-(3,3-dimethylureido)-2-phenylacetic acid was isolated as a white solid (0.59 g, 86%) and used without further purification. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.82 (s, 6H) 5.22 (d, J=7.32 Hz, 1H) 6.58 (d, J=7.32 Hz, 1H) 7.28 (t, J=7.17 Hz, 1H) 7.33 (t, J=7.32 Hz, 2H) 7.38-7.43 (m, 2H) 12.65 (s, 1H). LCMS: Anal. Calcd. for C₁₁H₁₄N₂O₃: 222.24; found: 223.21 (M+H)⁺. HPLC XTERRA C-18 3.0×50 mm, 0 to 100% B over 2 minutes, 1 minute hold time, A=90% water, 10% methanol, 0.2% H₃PO₄, B=10% water, 90% methanol, 0.2% H₃PO₄, RT=0.75 min, 93% homogeneity index.

Step 1; (R)-tert-butyl 2-(3-cyclopentylureido)-2-phenylacetate: To a stirred solution of (R)-2-amino-2-phenylacetic acid hydrochloride (1.0 g, 4.10 mmol) and Hunig's base (1.0 mL, 6.15 mmol) in DMF (15 mL) was added cyclopentyl isocyanate (0.46 mL, 4.10 mmol) dropwise and over 10 minutes. After stirring at room temperature for 3 hours, the reaction was concentrated under reduced pressure and the resulting residue was taken up in ethyl acetate. The organic layer was washed with H₂O and brine, dried (MgSO₄), filtered, and concentrated under reduced pressure. (R)-tert-butyl 2-(3-cyclopentylureido)-2-phenylacetate was obtained as an opaque oil (1.32 g; 100%) and used without further purification. NMR (500 MHz, CD₃Cl-D) δ ppm 1.50-1.57 (m, 2H) 1.58-1.66 (m, 2H) 1.87-1.97 (m, 2H) 3.89-3.98 (m, 1H) 5.37 (s, 1H) 7.26-7.38 (m, 5H). LCMS: Anal. Calcd. for C₁₈H₂₆N₂O₃ 318.19 found 319.21 (M+H)⁺; HPLC XTERRA C-18 3.0×50 mm, 0 to 100% B over 4 minutes, 1 minute hold time, A=90% water, 10% methanol, 0.1% TFA, B=10% water, 90% methanol, 0.1% TFA, RT=2.82 min, 96% homogeneity index.

Step 2; (R)-2-(3-cyclopentylureido)-2-phenylacetic acid: To a stirred solution of (R)-tert-butyl 2-(3-cyclopentylureido)-2-phenylacetate (1.31 g, 4.10 mmol) in CH₂Cl₂ (25 mL) was added TFA (4 mL) and triethylsilane (1.64 mL; 10.3 mmol) dropwise, and the resulting solution was stirred at room temperature for 6 hours. The volatile components were removed under reduced pressure and the crude product was recrystallized in ethyl acetate/pentanes to yield (R)-2-(3-cyclopentylureido)-2-phenylacetic acid as a white solid (0.69 g, 64%). ¹H NMR (500 MHz, DMSO-d₆). δ ppm 1.17-1.35 (m, 2H) 1.42-1.52 (m, 2H) 1.53-1.64 (m, 2H) 1.67-1.80 (m, 2H) 3.75-3.89 (m, 1H) 5.17 (d, J=7.93 Hz, 1H) 6.12 (d, J=7.32 Hz, 1H) 6.48 (d, J=7.93 Hz, 1H) 7.24-7.40 (m, 5H) 12.73 (s, 1H). LCMS: Anal. Calcd. for C₁₄H₁₈N₂O₃: 262.31; found: 263.15 (M+H)⁺. HPLC XTERRA C-18 3.0×50 mm, 0 to 100% B over 2 minutes, 1 minute hold time, A=90% water, 10% methanol, 0.2% H₃PO₄, B=10% water, 90% methanol, 0.2% H₃PO₄, RT=1.24 min, 100% homogeneity index.

To a stirred solution of 2-(benzylamino)acetic acid (2.0 g, 12.1 mmol) in formic acid (91 mL) was added formaldehyde (6.94 mL, 93.2 mmol). After five hours at 70° C., the reaction mixture was concentrated under reduced pressure to 20 mL and a white solid precipitated. Following filtration, the mother liquors were collected and further concentrated under reduced pressure providing the crude product. Purification by reverse-phase preparative HPLC (Xterra 30×100 mm, detection at 220 nm, flow rate 35 mL/min, 0 to 35% B over 8 min; A=90% water, 10% methanol, 0.1% TFA, B=10% water, 90% methanol, 0.1% TFA) provided the title compound 2-(benzyl(methyl)-amino)acetic acid as its TFA salt (723 mg, 33%) as a colorless wax. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 2.75 (s, 3H) 4.04 (s, 2H) 4.34 (s, 2H) 7.29-7.68 (m, 5H). LCMS: Anal. Calcd. for: C₁₀H₁₃NO₂ 175.05; Found: 180.20 (M+H)⁺.

To a stirred solution of 3-methyl-2-(methylamino)butanoic acid (0.50 g, 3.81 mmol) in water (30 mL) was added K₂CO₃ (2.63 g, 19.1 mmol) and benzyl chloride (1.32 g, 11.4 mmol). The reaction mixture was stirred at ambient temperature for 18 hours. The reaction mixture was extracted with ethyl acetate (30 mL×2) and the aqueous layer was concentrated under reduced pressure providing the crude product which was purified by reverse-phase preparative HPLC (Xterra 30×100 mm, detection at 220 nm, flow rate 40 mL/min, 20 to 80% B over 6 min; A=90% water, 10% methanol, 0.1% TFA, B=10% water, 90% methanol, 0.1% TFA) to provide 2-(benzyl(methyl)amino)-3-methylbutanoic acid, TFA salt (126 mg, 19%) as a colorless wax. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.98 (d, 3H) 1.07 (d, 3H) 2.33-2.48 (m, 1H) 2.54-2.78 (m, 3H) 3.69 (s, 1H) 4.24 (s, 2H) 7.29-7.65 (m, 5H). LCMS: Anal. Calcd. for: C₁₃H₁₉NO₂ 221.14; Found: 222.28 (M+H)⁺.

Na₂CO₃ (1.83 g, 17.2 mmol) was added to NaOH (33 mL of 1M/H₂O, 33 mmol) solution of L-valine (3.9 g, 33.29 mmol) and the resulting solution was cooled with ice-water bath. Methyl chloroformate (2.8 mL, 36.1 mmol) was added dropwise over 15 min, the cooling bath was removed and the reaction mixture was stirred at ambient temperature for 3.25 hr. The reaction mixture was washed with ether (50 mL, 3×), and the aqueous phase was cooled with ice-water bath and acidified with concentrated HCl to a pH region of 1-2, and extracted with CH₂Cl₂ (50 mL, 3×). The organic phase was dried (MgSO₄) and evaporated in vacuo to afford Cap-51 as a white solid (6 g). ¹H NMR for the dominant rotamer (DMSO-d₆, δ=2.5 ppm, 500 MHz): 12.54 (s, 1H), 7.33 (d, J=8.6, 1H), 3.84 (dd, J=8.4, 6.0, 1H), 3.54 (s, 3H), 2.03 (m, 1H), 0.87 (m, 6H). HRMS: Anal. Calcd. for [M+H]⁺ C₇H₁₄NO₄: 176.0923; found 176.0922.

Cap-52 was synthesized from L-alanine according to the procedure described for the synthesis of Cap-51. For characterization purposes, a portion of the crude material was purified by a reverse phase HPLC (H₂O/methanol/TFA) to afford Cap-52 as a colorless viscous oil. ¹H NMR (DMSO-d₆, δ5=2.5 ppm, 500 MHz): 12.49 (br s, 1H), 7.43 (d, J=7.3, 0.88H), 7.09 (app br s, 0.12H), 3.97 (m, 1H), 3.53 (s, 3H), 1.25 (d, J=7.3, 3H).

Caps-53 to -64 were prepared from appropriate starting materials according to the procedure described for the synthesis of Cap-51, with noted modifications if any

Cap Structure Analytical Data Cap-53a: (R) Cap-53b: (S)

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 500 MHz): δ 12.51 (br s, 1H), 7.4 (d, J = 7.9, 0.9H), 7.06 (app s, 0.1H), 3.86-3.82 (m, 1H), 3.53 (s, 3H), 1.75-1.67 (m, 1H), 1.62-1.54 (m, 1H), 0.88 (d, J = 7.3, 3H). RT = 0.77 minutes (Cond. 2); LC/MS: Anal. Calcd. for [M + Na]⁺ C₆H₁₁NNaO₄: 184.06; found 184.07. HRMS Calcd. for [M + Na]⁺ C₆H₁₁NNaO₄: 184.0586; found 184.0592. Cap-54a: (R) Cap-54b: (S)

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 500 MHz): δ 12.48 (s, 1H), 7.58 (d, J = 7.6, 0.9H), 7.25 (app s, 0.1H), 3.52 (s, 3H), 3.36-3.33 (m, 1H), 1.10-1.01 (m, 1H), 0.54-0.49 (m, 1H), 0.46-0.40 (m, 1H), 0.39-0.35 (m, 1H), 0.31-0.21 (m, 1H). HRMS Calcd. for [M + H]⁺ C₇H₁₂NO₄: 174.0766; found 174.0771 Cap-55

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 500 MHz): δ 12.62 (s, 1H), 7.42 (d, J = 8.2, 0.9H), 7.07 (app s, 0.1H), 5.80-5.72 (m, 1H), 5.10 (d, J = 17.1, 1H), 5.04 (d, J = 10.4, 1H), 4.01-3.96 (m, 1H), 3.53 (s, 3H), 2.47-2.42 (m, 1H), 2.35-2.29 (m, 1H). Cap-56

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 500 MHz): δ 12.75 (s, 1H), 7.38 (d, J = 8.3, 0.9H), 6.96 (app s, 0.1H), 4.20-4.16 (m, 1H), 3.60-3.55 (m, 2H), 3.54 (s, 3H) , 3.24 (s, 3H). Cap-57

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 500 MHz): δ 12.50 (s, 1H), 8.02 (d, J = 7.7, 0.9H), 7.40 (d, J = 7.9, 0.76H), 7.19 (d, J = 8.2, 0.7H), 7.07 (d, J = 6.7, 0.9H), 4.21-4.12 (m, 0.08H), 4.06-3.97 (m, 0.07H), 3.96-3.80 (m, 0.85H), 3.53 (s, 3H), 1.69-1.51 (m, 2H), 1.39-1.26 (m, 2H), 0.85 (t, J = 7.4, 3H). LC (Cond. 2): RT = 1.39 LC/MS: Anal. Calcd. for [M + H]⁺ C₇H₁₄NO₄: 176.09; found 176.06. Cap-58

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 500 MHz): δ 12.63 (br s, 1H), 7.35 (s, 1H), 7.31 (d, J = 8.2, 1H), 6.92 (s, 1H), 4.33-4.29 (m, 1H), 3.54 (s, 3H), 2.54 (dd, J = 15.5, 5.4, 1H), 2.45 (dd, J = 15.6, 8.0, 1H). RT = 0.16 (Cond. 2); LC/MS: Anal. Calcd. for [M + H]⁺ C₆H₁₁N₂O₅: 191.07; found 191.14. Cap-59a: (R) Cap-59b: (S)

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 400 MHz): δ 12.49 (br s, 1H), 7.40 (d, J = 7.3, 0.89H), 7.04 (br s, 0.11H), 4.00-3.95 (m, 3H), 1.24 (d, J = 7.3, 3H), 1.15 (t, J = 7.2, 3H). HRMS: Anal. Calcd. for [M + H]⁺ C₆H₁₂NO₄: 162.0766; found 162.0771. Cap-60

The crude material was purified with a reverse phase HPLC (H₂O/MeOH/TFA) to afford a colorless viscous oil that crystallized to a white solid upon exposure to high vacuum. ¹H NMR (DMSO-d₆, δ = 2.5 ppm, 400 MHz): δ 12.38 (br s, 1H), 7.74 (s, 0.82H), 7.48 (s, 0.18H), 3.54/3.51 (two s, 3H), 1.30 (m, 2H), 0.98 (m, 2H). HRMS: Anal. Calcd. for [M + H]⁺ C₆H₁₀NO₄: 160.0610; found 160.0604. Cap-61

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 400 MHz): δ 12.27 (br s, 1H), 7.40 (br s, 1H), 3.50 (s, 3H), 1.32 (s, 6H). HRMS: Anal. Calcd. for [M + H]⁺ C₆H₁₂NO₄: 162.0766; found 162.0765. Cap-62

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 400 MHz): δ 12.74 (br s, 1H), 4.21 (d, J = 10.3, 0.6H), 4.05 (d, J = 10.0, 0.4H), 3.62/3.60 (two singlets, 3H), 3.0 (s, 3H), 2.14-2.05 (m, 1H), 0.95 (d, J = 6.3, 3H), 0.81 (d, J = 6.6, 3H). LC/MS Anal. Calcd. for [M − H]⁻ C₈H₁₄NO₄: 188.09; found 188.05. Cap-63

[Note: the reaction was allowed to run for longer than what was noted for the general procedure.] ¹H NMR (DMSO-d₆, δ = 2.5 ppm, 400 MHz): 12.21 (br s, 1H), 7.42 (br s, 1H), 3.50 (s, 3H), 2.02-1.85 (m, 4H), 1.66-1.58 (m, 4H). LC/MS: Anal. Calcd. for [M + H]⁺ C₈H₁₄NO₄: 188.09; found 188.19. Cap-64

[Note: the reaction was allowed to run for longer than what was noted for the general procedure.] ¹H NMR (DMSO-d₆, δ = 2.5 ppm, 400 MHz): 12.35 (br s, 1H), 7.77 (s, 0.82H), 7.56/7.52 (overlapping br s, 0.18H), 3.50 (s, 3H), 2.47-2.40 (m, 2H0, 2.14-2.07 (m, 2H), 1.93-1.82 (m, 2H).

Methyl chloroformate (0.65 mL, 8.39 mmol) was added dropwise over 5 min to a cooled (ice-water) mixture of Na₂CO₃ (0.449 g, 4.23 mmol), NaOH (8.2 mL of 1M/H₂O, 8.2 mmol) and (S)-2-amino-3-hydroxy-3-methylbutanoic acid (1.04 g, 7.81 mmol). The reaction mixture was stirred for 45 min, and then the cooling bath was removed and stirring was continued for an additional 3.75 hr. The reaction mixture was washed with CH₂Cl₂, and the aqueous phase was cooled with ice-water bath and acidified with concentrated HCl to a pH region of 1-2. The volatile component was removed in vacuo and the residue was taken up in a 2:1 mixture of MeOH/CH₂Cl₂ (15 mL) and filtered, and the filterate was rotervaped to afford Cap-65 as a white semi-viscous foam (1.236 g). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): δ 6.94 (d, J=8.5, 0.9 H), 6.53 (br s, 0.1H), 3.89 (d, J=8.8, 1H), 2.94 (s, 3H), 1.15 (s, 3H), 1.13 (s, 3H).

Cap-66 and -67 were prepared from appropriate commercially available starting materials by employing the procedure described for the synthesis of Cap-65.

¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): δ 12.58 (br s, 1H), 7.07 (d, J=8.3, 0.13H), 6.81 (d, J=8.8, 0.67H), 4.10-4.02 (m, 1.15H), 3.91 (dd, J=9.1, 3.5, 0.85H), 3.56 (s, 3H), 1.09 (d, J=6.2, 3H). [Note: only the dominant signals of NH were noted].

¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): 12.51 (br s, 1H), 7.25 (d, J=8.4, 0.75H), 7.12 (br d, J=0.4, 0.05H), 6.86 (br s, 0.08H), 3.95-3.85 (m, 2H), 3.54 (s, 3H), 1.08 (d, J=6.3, 3H). [Note: only the dominant signals of NH were noted].

Methyl chloroformate (0.38 ml, 4.9 mmol) was added drop-wise to a mixture of 1N NaOH (aq) (9.0 ml, 9.0 mmol), 1M NaHCO₃ (aq) (9.0 ml, 9.0 mol), L-aspartic acid β-benzyl ester (1.0 g, 4.5 mmol) and Dioxane (9 ml). The reaction mixture was stirred at ambient conditions for 3 hr, and then washed with Ethyl acetate (50 ml, 3×). The aqueous layer was acidified with 12N HCl to a pH ˜1-2, and extracted with ethyl acetate (3×50 ml). The combined organic layers were washed with brine, dried (Na₂SO₄), filtered, and concentrated in vacuo to afford Cap-68 as a light yellow oil (1.37 g; mass is above theoretical yield, and the product was used without further purification). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 500 MHz): δ 12.88 (br s, 1H), 7.55 (d, J=8.5, 1H), 7.40-7.32 (m, 5H), 5.13 (d, J=12.8, 1H), 5.10 (d, J=12.9, 1H), 4.42-4.38 (m, 1H), 3.55 (s, 3H), 2.87 (dd, J=16.2, 5.5, 1H), 2.71 (dd, J=16.2, 8.3, 1H). LC (Cond. 2): RT=1.90 min; LC/MS: Anal. Calcd. For [M+H]⁺ C₁₃H₁₆NO₆: 282.10; found 282.12.

NaCNBH₃ (2.416 g, 36.5 mmol) was added in batches to a chilled (˜15° C.) water (17 mL)/MeOH (10 mL) solution of alanine (1.338 g, 15.0 mmol). A few minutes later acetaldehyde (4.0 mL, 71.3 mmol) was added drop-wise over 4 min, the cooling bath was removed, and the reaction mixture was stirred at ambient condition for 6 hr. An additional acetaldehyde (4.0 mL) was added and the reaction was stirred for 2 hr. Concentrated HCl was added slowly to the reaction mixture until the pH reached ˜1.5, and the resulting mixture was heated for 1 hr at 40° C. Most of the volatile component was removed in vacuo and the residue was purified with a Dowex® 50WX8-100 ion-exchange resin (column was washed with water, and the compound was eluted with dilute NH₄OH, prepared by mixing 18 ml of NH₄OH and 282 ml of water) to afford Cap-69 (2.0 g) as an off-white soft hygroscopic solid. ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): δ 3.44 (q, J=7.1, 1H), 2.99-2.90 (m, 2H), 2.89-2.80 (m, 2H), 1.23 (d, J=7.1, 3H), 1.13 (t, J=7.3, 6H).

Cap-70 to -74x were prepared according to the procedure described for the synthesis of Cap-69 by employing appropriate starting materials.

Cap Structure Analytical Data Cap-71a: (R) Cap-71b: (S)

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 500 MHz): δ 3.18-3.14 (m, 1H), 2.84-2.77 (m, 2H), 2.76-2.68 (m, 2H), 1.69-1.54 (m, 2H), 1.05 (t, J = 7.2, 6H), 0.91 (t, J = 7.3, 3H). LC/MS: Anal. Calcd. for [M + H]⁺ C₈H₁₈NO₂: 160.13; found 160.06. Cap-72

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 400 MHz): δ 2.77-2.66 (m, 3H), 2.39-2.31 (m, 2H), 1.94-1.85 (m, 1H), 0.98 (t, J = 7.1, 6H), 0.91 (d, J = 6.5, 3H), 0.85 (d, J = 6.5, 3H). LC/MS: Anal. Calcd. for [M + H]⁺ C₉H₂₀NO₂: 174.15; found 174.15. Cap-73

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 500 MHz): δ 9.5 (br s, 1H), 3.77 (dd, J = 10.8, 4.1, 1H), 3.69-3.61 (m, 2H), 3.26 (s, 3H), 2.99-2.88 (m, 4H), 1.13 (t, J = 7.2, 6H). Cap-74

¹H NMR (DMSO-d₆, δ = 2.5 ppm, 500 MHz): δ 7.54 (s, 1H), 6.89 (s, 1H), 3.81 (t, J = 6.6, k, 1H), 2.82-2.71 (m, 4H), 2.63 (dd, J = 15.6, 7.0, 1H), 2.36 (dd, J = 15.4, 6.3, 1H), 1.09 (t, J = 7.2, 6H). RT = 0.125 minutes (Cond. 2); LC/MS: Anal. Calcd. for [M + H]⁺ C₈H₁₇N₂O₃: 189.12; found 189.13. Cap-74x

LC/MS: Anal. Calcd. for [M + H]⁺ C₁₀H₂₂NO₂: 188.17; found 188.21

NaBH₃CN (1.6 g, 25.5 mmol) was added to a cooled (ice/water bath) water (25 ml)/methanol (15 ml) solution of H-D-Ser-OBzl HCl (2.0 g, 8.6 mmol). Acetaldehyde (1.5 ml, 12.5 mmol) was added drop-wise over 5 min, the cooling bath was removed, and the reaction mixture was stirred at ambient condition for 2 hr. The reaction was carefully quenched with 12N HCl and concentrated in vacuo. The residue was dissolved in water and purified with a reverse phase HPLC (MeOH/H₂O/TFA) to afford the TFA salt of (R)-benzyl 2-(diethylamino)-3-hydroxypropanoate as a colorless viscous oil (1.9 g). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 500 MHz): δ 9.73 (br s, 1H), 7.52-7.36 (m, 5H), 5.32 (d, J=12.2, 1H), 5.27 (d, J=12.5, 1H), 4.54-4.32 (m, 1H), 4.05-3.97 (m, 2H), 3.43-3.21 (m, 4H), 1.23 (t, J=7.2, 6H). LC/MS (Cond. 2): RT=1.38 min; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₄H₂₂NO₃: 252.16; found 252.19.

Cap-75

NaH (0.0727 g, 1.82 mmol, 60%) was added to a cooled (ice-water) THF (3.0 mL) solution of the TFA salt (R)-benzyl 2-(diethylamino)-3-hydroxypropanoate (0.3019 g, 0.8264 mmol) prepared above, and the mixture was stirred for 15 min. Methyl iodide (56 μL, 0.90 mmol) was added and stirring was continued for 18 hr while allowing the bath to thaw to ambient condition. The reaction was quenched with water and loaded onto a MeOH pre-conditioned MCX (6 g) cartridge, and washed with methanol followed by compound elution with 2N NH₃/Methanol. Removal of the volatile component in vacuo afforded Cap-75, contaminated with (R)-2-(diethylamino)-3-hydroxypropanoic acid, as a yellow semi-solid (100 mg). The product was used as is without further purification.

NaCNBH₃ (1.60 g, 24.2 mmol) was added in batches to a chilled (˜15° C.) water/MeOH (12 mL each) solution of (S)-4-amino-2-(tent-butoxycarbonylamino) butanoic acid (2.17 g, 9.94 mmol). A few minutes later acetaldehyde (2.7 mL, 48.1 mmol) was added drop-wise over 2 min, the cooling bath was removed, and the reaction mixture was stirred at ambient condition for 3.5 hr. An additional acetaldehyde (2.7 mL, 48.1 mmol) was added and the reaction was stirred for 20.5 hr. Most of the MeOH component was removed in vacuo, and the remaining mixture was treated with concentrated HCl until its pH reached ˜1.0 and then heated for 2 hr at 40° C. The volatile component was removed in vacuo, and the residue was treated with 4 M HCl/dioxane (20 mL) and stirred at ambient condition for 7.5 hr. The volatile component was removed in vacuo and the residue was purified with Dowex® 50WX8-100 ion-exchange resin (column was washed with water and the compound was eluted with dilute NH₄OH, prepared from 18 ml of NH₄OH and 282 ml of water) to afford intermediate (S)-2-amino-4-(diethylamino)butanoic acid as an off-white solid (1.73 g).

Methyl chloroformate (0.36 mL, 4.65 mmol) was added drop-wise over 11 min to a cooled (ice-water) mixture of Na₂CO₃ (0.243 g, 2.29 mmol), NaOH (4.6 mL of 1M/H₂O, 4.6 mmol) and the above product (802.4 mg). The reaction mixture was stirred for 55 min, and then the cooling bath was removed and stirring was continued for an additional 5.25 hr. The reaction mixture was diluted with equal volume of water and washed with CH₂Cl₂ (30 mL, 2×), and the aqueous phase was cooled with ice-water bath and acidified with concentrated HCl to a pH region of 2. The volatile component was then removed in vacuo and the crude material was free-based with MCX resin (6.0 g; column was washed with water, and sample was eluted with 2.0 M NH₃/MeOH) to afford impure Cap-76 as an off-white solid (704 mg). ¹H NMR (MeOH-d₄, δ=3.29 ppm, 400 MHz): δ 3.99 (dd, J=7.5, 4.7, 1H), 3.62 (s, 3H), 3.25-3.06 (m, 6H), 2.18-2.09 (m, 1H), 2.04-1.96 (m, 1H), 1.28 (t, J=7.3, 6H). LC/MS: Anal. Calcd. for [M+H]⁺ C₁₀H₂₁N₂O₄: 233.15; found 233.24.

The synthesis of Cap-77 was conducted according to the procedure described for Cap-7 by using 7-azabicyclo[2.2.1]heptane for the SN₂ displacement step, and by effecting the enantiomeric separation of the intermediate benzyl 2-(7-azabicyclo[2.2.1]heptan-7-yl)-2-phenylacetate using the following condition: the intermediate (303.7 mg) was dissolved in ethanol, and the resulting solution was injected on a chiral HPLC column (Chiracel AD-H column, 30×250 mm, 5 um) eluting with 90% CO₂—10% EtOH at 70 mL/min, and a temperature of 35° C. to provide 124.5 mg of enantiomer-1 and 133.8 mg of enantiomer-2. These benzyl esters were hydrogenolysed according to the preparation of Cap-7 to provide Cap-77: ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): δ 7.55 (m, 2H), 7.38-7.30 (m, 3H), 4.16 (s, 1H), 3.54 (app br s, 2H), 2.08-1.88 (m, 4 H), 1.57-1.46 (m, 4H). LC (Cond. 1): RT=0.67 min; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₄H₁₈NO₂: 232.13; found 232.18. HRMS: Anal. Calcd. for [M+H]⁺ C₁₄H₁₈NO₂: 232.1338; found 232.1340.

NaCNBH₃ (0.5828 g, 9.27 mmol) was added to a mixture of the HCl salt of (R)-2-(ethylamino)-2-phenylacetic acid (an intermediate in the synthesis of Cap-3; 0.9923 mg, 4.60 mmol) and (1-ethoxycyclopropoxy)trimethylsilane (1.640 g, 9.40 mmol) in MeOH (10 mL), and the semi-heterogeneous mixture was heated at 50° C. with an oil bath for 20 hr. More (1-ethoxycyclopropoxy)trimethylsilane (150 mg, 0.86 mmol) and NaCNBH₃ (52 mg, 0.827 mmol) were added and the reaction mixture was heated for an additional 3.5 hr. It was then allowed to cool to ambient temperature and acidified to a ˜pH region of 2 with concentrated HCl, and the mixture was filtered and the filtrate was rotervaped. The resulting crude material was taken up in i-PrOH (6 mL) and heated to effect dissolution, and the non-dissolved part was filtered off and the filtrate concentrated in vacuo. About ⅓ of the resultant crude material was purified with a reverse phase HPLC (H₂O/MeOH/TFA) to afford the TFA salt of Cap-78 as a colorless viscous oil (353 mg). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz; after D₂O exchange): δ 7.56-7.49 (m, 5H), 5.35 (S, 1H), 3.35 (m, 1H), 3.06 (app br s, 1H), 2.66 (m, 1H), 1.26 (t, J=7.3, 3H), 0.92 (m, 1H), 0.83-0.44 (m, 3H). LC (Cond. 1): RT=0.64 min; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₃H₁₈NO₂: 220.13; found 220.21. HRMS: Anal. Calcd. for [M+]⁺ C₁₃H₁₈NO₂: 220.1338; found 220.1343.

Ozone was bubbled through a cooled (−78° C.) CH₂Cl₂ (5.0 mL) solution Cap-55 (369 mg, 2.13 mmol) for about 50 min until the reaction mixture attained a tint of blue color. Me₂S (10 pipet drops) was added, and the reaction mixture was stirred for 35 min. The −78° C. bath was replaced with a −10° C. bath and stirring continued for an additional 30 min, and then the volatile component was removed in vacuo to afford a colorless viscous oil.

NaBH₃CN (149 mg, 2.25 mmol) was added to a MeOH (5.0 mL) solution of the above crude material and morpholine (500 μL, 5.72 mmol) and the mixture was stirred at ambient condition for 4 hr. It was cooled to ice-water temperature and treated with concentrated HCl to bring its pH to ˜2.0, and then stirred for 2.5 hr. The volatile component was removed in vacuo, and the residue was purified with a combination of MCX resin (MeOH wash; 2.0 N NH₃/MeOH elution) and a reverse phase HPLC (H₂O/MeOH/TFA) to afford Cap-79 containing unknown amount of morpholine.

In order to consume the morpholine contaminant, the above material was dissolved in CH₂Cl₂ (1.5 mL) and treated with Et₃N (0.27 mL, 1.94 mmol) followed by acetic anhydride (0.10 mL, 1.06 mmol) and stirred at ambient condition for 18 hr. THF (1.0 mL) and H₂O (0.5 mL) were added and stirring continued for 1.5 hr. The volatile component was removed in vacuo, and the resultant residue was passed through MCX resin (MeOH wash; 2.0 N NH₃/MeOH elution) to afford impure Cap-79 as a brown viscous oil, which was used for the next step without further purification.

SOCl₂ (6.60 mL, 90.5 mmol) was added drop-wise over 15 min to a cooled (ice-water) mixture of (S)-3-amino-4-(benzyloxy)-4-oxobutanoic acid (10.04 g, 44.98 mmol) and MeOH (300 mL), the cooling bath was removed and the reaction mixture was stirred at ambient condition for 29 hr. Most of the volatile component was removed in vacuo and the residue was carefully partitioned between EtOAc (150 mL) and saturated NaHCO₃ solution. The aqueous phase was extracted with EtOAc (150 mL, 2×), and the combined organic phase was dried (MgSO₄), filtered, and concentrated in vacuo to afford (S)-1-benzyl 4-methyl 2-aminosuccinate as a colorless oil (9.706 g). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): δ 7.40-7.32 (m, 5H), 5.11 (s, 2H), 3.72 (app t, J=6.6, 1H), 3.55 (s, 3H), 2.68 (dd, J=15.9, 6.3, 1H), 2.58 (dd, J=15.9, 6.8, 1H), 1.96 (s, 2H). LC (Cond. 1): RT=0.90 min; LC/MS: Anal. Calcd. for [M+H]⁺ C₁₂H₁₆NO₄: 238.11; found 238.22.

Pb(NO₃)₂ (6.06 g, 18.3 mmol) was added over 1 min to a CH₂Cl₂ (80 mL) solution of (S)-1-benzyl 4-methyl 2-aminosuccinate (4.50 g, 19.0 mmol), 9-bromo-9-phenyl-9H-fluorene (6.44 g, 20.0 mmol) and Et₃N (3.0 mL, 21.5 mmol), and the heterogeneous mixture was stirred at ambient condition for 48 hr. The mixture was filtered and the filtrate was treated with MgSO₄ and filtered again, and the final filtrate was concentrated. The resulting crude material was submitted to a Biotage purification (350 g silica gel, CH₂Cl₂ elution) to afford (S)-1-benzyl 4-methyl 2-(9-phenyl-9H-fluoren-9-ylamino)succinate as highly viscous colorless oil (7.93 g). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): δ 7.82 (m, 2H), 7.39-7.13 (m, 16H), 4.71 (d, J=12.4, 1H), 4.51 (d, J=12.6, 1H), 3.78 (d, J=9.1, NH), 3.50 (s, 3H), 2.99 (m, 1H), 2.50-2.41 (m, 2H, partially overlapped with solvent). LC (Cond. 1): RT=2.16 min; LC/MS: Anal. Calcd. for [M+H]⁺ C₃₁H₂₈NO₄: 478.20; found 478.19.

LiHMDS (9.2 mL of 1.0 M/THF, 9.2 mmol) was added drop-wise over 10 min to a cooled (−78° C.) THF (50 mL) solution of (S)-1-benzyl 4-methyl 2-(9-phenyl-9H-fluoren-9-ylamino)succinate (3.907 g, 8.18 mmol) and stirred for ˜1 hr. MeI (0.57 mL, 9.2 mmol) was added drop-wise over 8 min to the mixture, and stirring was continued for 16.5 hr while allowing the cooling bath to thaw to room temperature. After quenching with saturated NH₄Cl solution (5 mL), most of the organic component was removed in vacuo and the residue was partitioned between CH₂Cl₂ (100 mL) and water (40 mL). The organic layer was dried (MgSO₄), filtered, and concentrated in vacuo, and the resulting crude material was purified with a Biotage (350 g silica gel; 25% EtOAc/hexanes) to afford 3.65 g of a 2S/3S and 2S/3R diastereomeric mixtures of 1-benzyl 4-methyl 3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)succinate in ˜1.0:0.65 ratio (¹H NMR). The stereochemistry of the dominant isomer was not determined at this juncture, and the mixture was submitted to the next step without separation. Partial ¹H NMR data (DMSO-d₆, δ=2.5 ppm, 400 MHz): major diastereomer, δ 4.39 (d, J=12.3, 1H of CH₂), 3.33 (s, 3H, overlapped with H₂O signal), 3.50 (d, J=10.9, NH), 1.13 (d, J=7.1, 3H); minor diastereomer, δ 4.27 (d, J=12.3, 1H of CH₂), 3.76 (d, J=10.9, NH), 3.64 (s, 3H), 0.77 (d, J=7.0, 3H). LC (Cond. 1): RT=2.19 min; LC/MS: Anal. Calcd. for [M+H]⁺ C₃₂H₃₀NO₄: 492.22; found 492.15.

Diisobutylaluminum hydride (20.57 ml of 1.0 M in hexanes, 20.57 mmol) was added drop-wise over 10 min to a cooled (−78° C.) THF (120 mL) solution of (2S)-1-benzyl 4-methyl 3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)succinate (3.37 g, 6.86 mmol) prepared above, and stirred at −78° C. for 20 hr. The reaction mixture was removed from the cooling bath and rapidly poured into ˜1M H₃PO₄/H₂O (250 mL) with stirring, and the mixture was extracted with ether (100 mL, 2×). The combined organic phase was washed with brine, dried (MgSO₄), filtered and concentrated in vacuo. A silica gel mesh of the crude material was prepared and submitted to chromatography (25% EtOAc/hexanes; gravity elution) to afford 1.1 g of (2S,3S)-benzyl 4-hydroxy-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate, contaminated with benzyl alcohol, as a colorless viscous oil and (2S,3R)-benzyl 4-hydroxy-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)-butanoate containing the (2S,3R) stereoisomer as an impurity. The later sample was resubmitted to the same column chromatography purification conditions to afford 750 mg of purified material as a white foam. [Note: the (2S, 3S) isomer elutes before the (2S,3R) isomer under the above condition]. (2S,3S) isomer: ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): 7.81 (m, 2H), 7.39-7.08 (m, 16H), 4.67 (d, J=12.3, 1H), 4.43 (d, J=12.4, 1H), 4.21 (app t, J=5.2, OH), 3.22 (d, J=10.1, NH), 3.17 (m, 1H), 3.08 (m, 1H), ˜2.5 (m, 1H, overlapped with the solvent signal), 1.58 (m, 1H), 0.88 (d, J=6.8, 3H). LC (Cond. 1): RT=2.00 min; LC/MS: Anal. Calcd. for [M+H]⁺ C₃₁H₃₀NO₃: 464.45; found 464.22. (2S,3R) isomer: ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): 7.81 (d, J=7.5, 2H), 7.39-7.10 (m, 16H), 4.63 (d, J=12.1, 1H), 4.50 (app t, J=4.9, 1H), 4.32 (d, J=12.1, 1H), 3.59-3.53 (m, 2H), 3.23 (m, 1H), 2.44 (dd, J=9.0, 8.3, 1H), 1.70 (m, 1H), 0.57 (d, J=6.8, 3H). LC (Cond. 1): RT=1.92 min; LC/MS: Anal. Calcd. for [M+H]⁺ C₃₁H₃₀NO₃: 464.45; found 464.52.

The relative stereochemical assignments of the DIBAL-reduction products were made based on NOE studies conducted on lactone derivatives prepared from each isomer by employing the following protocol: LiHMDS (50 μL of 1.0 M/THF, 0.05 mmol) was added to a cooled (ice-water) THF (2.0 mL) solution of (2S,3S)-benzyl 4-hydroxy-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)-butanoate (62.7 mg, 0.135 mmol), and the reaction mixture was stirred at similar temperature for ˜2 hr. The volatile component was removed in vacuo and the residue was partitioned between CH₂Cl₂ (30 mL), water (20 mL) and saturated aqueous NH₄Cl solution (1 mL). The organic layer was dried (MgSO₄), filtered, and concentrated in vacuo, and the resulting crude material was submitted to a Biotage purification (40 g silica gel; 10-15% EtOAc/hexanes) to afford (3S,4S)-4-methyl-3-(9-phenyl-9H-fluoren-9-ylamino)dihydrofuran-2(3H)-one as a colorless film of solid (28.1 mg). (2S,3R)-benzyl 4-hydroxy-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate was elaborated similarly to (3S,4R)-4-methyl-3-(9-phenyl-9H-fluoren-9-ylamino)dihydrofuran-2(3H)-one. (3S,4S)-lactone isomer: ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz), 7.83 (d, J=7.5, 2H), 7.46-7.17 (m, 11H), 4.14 (app t, J=8.3, 1H), 3.60 (d, J=5.8, NH), 3.45 (app t, J=9.2, 1H), ˜2.47 (m, 1H, partially overlapped with solvent signal), 2.16 (m, 1H), 0.27 (d, J=6.6, 3H). LC (Cond. I): RT=1.98 min; LC/MS: Anal. Calcd. for [M+Na]⁺ C₂₄H₂₁NNaO₂: 378.15; found 378.42. (3S,4R)-lactone isomer: ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz), 7.89 (d, J=7.6, 1H), 7.85 (d, J=7.3, 1H), 7.46-7.20 (m, 11H), 3.95 (dd, J=9.1, 4.8, 1H), 3.76 (d, J=8.8, 1H), 2.96 (d, J=3.0, NH), 2.92 (dd, J=6.8, 3, NCH), 1.55 (m, 1H), 0.97 (d, J=7.0, 3H). LC (Cond. 1): RT=2.03 min; LC/MS: Anal. Calcd. for [M+Na]⁺ C₂₄H₂₁NNaO₂: 378.15; found 378.49.

BDMS-Cl (48 mg, 0.312 mmol) followed by imidazole (28.8 mg, 0.423 mmol) were added to a CH₂Cl₂ (3 ml) solution of (2S,3S)-benzyl 4-hydroxy-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate (119.5 mg, 0.258 mmol), and the mixture was stirred at ambient condition for 14.25 hr. The reaction mixture was then diluted with CH₂Cl₂ (30 mL) and washed with water (15 mL), and the organic layer was dried (MgSO₄), filtered, and concentrated in vacuo. The resultant crude material was purified with a Biotage (40 g silica gel; 5% EtOAc/hexanes) to afford (2S,3S)-benzyl 4-(tert-butyldimethylsilyloxy)-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate, contaminated with TBDMS based impurities, as a colorless viscous oil (124.4 mg). (2S,3R)-benzyl 4-hydroxy-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate was elaborated similarly to (2S,3R)-benzyl 4-(tert-butyldimethylsilyloxy)-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate. (2S,3S)-silyl ether isomer: ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz), 7.82 (d, J=4.1, 1H), 7.80 (d, J=4.0, 1H), 7.38-7.07 (m, 16 H), 4.70 (d, J=12.4, 1H), 4.42 (d, J=12.3, 1H), 3.28-3.19 (m, 3H), 2.56 (dd, J=10.1, 5.5, 1H), 1.61 (m, 1H), 0.90 (d, J=6.8, 3H), 0.70 (s, 9H), −0.13 (s, 3H), −0.16 (s, 3H). LC (Cond. 1, where the run time was extended to 4 min): RT=3.26 min; LC/MS: Anal. Calcd. for [M+H]⁺ C₃₇H₄₄NO₃Si: 578.31; found 578.40. (2S,3R)-silyl ether isomer: ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz), 7.82 (d, J=3.0, 1H), 7.80 (d, J=3.1, 1H), 7.39-7.10 (m, 16H), 4.66 (d, J=12.4, 1H), 4.39 (d, J=12.4, 1H), 3.61 (dd, J=9.9, 5.6, 1H), 3.45 (d, J=9.5, 1H), 3.41 (dd, J=10, 6.2, 1H), 2.55 (dd, J=9.5, 7.3, 1H), 1.74 (m, 1H), 0.77 (s, 9H), 0.61 (d, J=7.1, 3H), −0.06 (s, 3H), −0.08 (s, 3H).

A balloon of hydrogen was attached to a mixture of (2S,3S)-benzyl 4-(tert-butyldimethylsilyloxy)-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate (836 mg, 1.447 mmol) and 10% Pd/C (213 mg) in EtOAc (16 mL) and the mixture was stirred at room temperature for ˜21 hr, where the balloon was recharged with H₂ as necessary. The reaction mixture was diluted with CH₂Cl₂ and filtered through a pad of diatomaceous earth (Celite-545®), and the pad was washed with EtOAc (200 mL), EtOAc/MeOH (1:1 mixture, 200 mL) and MeOH (750 mL). The combined organic phase was concentrated, and a silica gel mesh was prepared from the resulting crude material and submitted to a flash chromatography (8:2:1 mixture of EtOAc/i-PrOH/H₂O) to afford (2S,3S)-2-amino-4-(tert-butyldimethylsilyloxy)-3-methylbutanoic acid as a white fluffy solid (325 mg). (2S,3R)-benzyl 4-(tert-butyldimethylsilyloxy)-3-methyl-2-(9-phenyl-9H-fluoren-9-ylamino)butanoate was similarly elaborated to (2S,3R)-2-amino-4-(tert-butyldimethylsilyloxy)-3-methylbutanoic acid. (2S,3S)-amino acid isomer: ¹H NMR (Methanol-d₄, δ=3.29 ppm, 400 MHz), 3.76 (dd, J=10.5, 5.2, 1H), 3.73 (d, J=3.0, 1H), 3.67 (dd, J=10.5, 7.0, 1H), 2.37 (m, 1H), 0.97 (d, J=7.0, 3H), 0.92 (s, 9H), 0.10 (s, 6H). LC/MS: Anal. Calcd. for [M+H]⁺ C₁₁H₂₆NO₃Si: 248.17; found 248.44. (2S,3R)-amino acid isomer: ¹H NMR (Methanol-d₄, δ=3.29 ppm, 400 MHz), 3.76-3.75 (m, 2H), 3.60 (d, J=4.1, 1H), 2.16 (m, 1H), 1.06 (d, J=7.3, 3H), 0.91 (s, 9H), 0.09 (s, 6H). Anal. Calcd. for [M+H]⁺ C₁₁H₂₆NO₃Si: 248.17; found 248.44.

Water (1 mL) and NaOH (0.18 mL of 1.0 M/H₂O, 0.18 mmol) were added to a mixture of (2S,3S)-2-amino-4-(tert-butyldimethylsilyloxy)-3-methylbutanoic acid (41.9 mg, 0.169 mmol) and Na₂CO₃ (11.9 mg, 0.112 mmol), and sonicated for about 1 min to effect dissolution of reactants. The mixture was then cooled with an ice-water bath, methyl chloroformate (0.02 mL, 0.259 mmol) was added over 30 s, and vigorous stirring was continued at similar temperature for 40 min and then at ambient temperature for 2.7 hr. The reaction mixture was diluted with water (5 mL), cooled with ice-water bath and treated drop-wise with 1.0 N HCl aqueous solution (˜0.23 mL). The mixture was further diluted with water (10 mL) and extracted with CH₂Cl₂ (15 mL, 2×). The combined organic phase was dried (MgSO₄), filtered, and concentrated in vacuo to afford Cap-80a as an off-white solid. (2S,3R)-2-amino-4-(tert-butyldimethylsilyloxy)-3-methylbutanoic acid was similarly elaborated to Cap-80b. Cap-80a: ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz), 12.57 (br s, 1H), 7.64 (d, J=8.3, 0.3H), 7.19 (d, J=8.8, 0.7H), 4.44 (dd, J=8.1, 4.6, 0.3H), 4.23 (dd, J=8.7, 4.4, 0.7H), 3.56/3.53 (two singlets, 3H), 3.48-3.40 (m, 2H), 2.22-2.10 (m, 1H), 0.85 (s, 9H), ˜0.84 (d, 0.9H, overlapped with t-Bu signal), 0.79 (d, J=7, 2.1H), 0.02/0.01/0.00 (three overlapping singlets, 6H). LC/MS: Anal. Calcd. for [M+Na]⁺ C₁₃H₂₇NNaO₅Si: 328.16; found 328.46. Cap-80b: ¹H NMR (CDCl₃, δ=7.24 ppm, 400 MHz), 6.00 (br d, J=6.8, 1H), 4.36 (dd, J=7.1, 3.1, 1H), 3.87 (dd, J=10.5, 3.0, 1H), 3.67 (s, 3H), 3.58 (dd, J=10.6, 4.8, 1H), 2.35 (m, 1H), 1.03 (d, J=7.1, 3H), 0.90 (s, 9H), 0.08 (s, 6H). LC/MS: Anal. Calcd. for [M+Na]⁺ C₁₃H₂₇NNaO₅Si: 328.16; found 328.53. The crude products were utilized without further purification.

Prepared according to the protocol described by Falb et al. Synthetic Communications 1993, 23, 2839.

Cap-82 to Cap-85

Cap-82 to Cap-85 were synthesized from appropriate starting materials according to the procedure described for Cap-51 or Cap-13. The samples exhibited similar spectral profiles as that of their enantiomers (i.e., Cap-4, Cap-13, Cap-51 and Cap-52, respectively).

To a mixture of 0-methyl-L-threonine (3.0 g, 22.55 mmol), NaOH (0.902 g, 22.55 mmol) in H₂O (15 mL) was added ClCO₂Me (1.74 mL, 22.55 mmol) dropwise at 0° C. The mixture was allowed to stir for 12 h and acidified to pH 1 using 1N HCl. The aqueous phase was extracted with EtOAc and (2×250 mL) and 10% MeOH in CH₂Cl₂ (250 mL) and the combined organic phases were concentrated under in vacuo to afford a colorless oil (4.18 g, 97%) which was of sufficient purity for use in subsequent steps. ¹HNMR (400 MHz, CDCl₃) δ 4.19 (s, 1H), 3.92-3.97 (m, 1H), 3.66 (s, 3H), 1.17 (d, J=7.7 Hz, 3H). LCMS: Anal. Calcd. for C₇H₁₃NO₅: 191; found: 190 (M−H)⁻.

To a mixture of L-homoserine (2.0 g, 9.79 mmol), Na₂CO₃ (2.08 g, 19.59 mmol) in H₂O (15 mL) was added ClCO₂Me (0.76 mL, 9.79 mmol) dropwise at 0° C. The mixture was allowed to stir for 48 h and acidified to pH 1 using 1N HCl. The aqueous phase was extracted with EtOAc and (2×250 mL) and the combined organic phases were concentrated under in vacuo to afford a colorless solid (0.719 g, 28%) which was of sufficient purity for use in subsequent steps. ¹HNMR (400 MHz, CDCl₃) δ 4.23 (dd, J=4.5, 9.1 Hz, 1H), 3.66 (s, 3H), 3.43-3.49 (m, 2H), 2.08-2.14 (m, 1H), 1.82-1.89 (m, 1H). LCMS: Anal. Calcd. for C₇H₁₃NO₅: 191; found: 192 (M+H)⁺.

A mixture of L-valine (1.0 g, 8.54 mmol), 3-bromopyridine (1.8 mL, 18.7 mmol), K₂CO₃ (2.45 g, 17.7 mmol) and CuI (169 mg, 0.887 mmol) in DMSO (10 mL) was heated at 100° C. for 12 h. The reaction mixture was cooled to rt, poured into H₂O (ca. 150 mL) and washed with EtOAc (×2). The organic layers were extracted with a small amount of H₂O and the combined aq phases were acidified to ca. pH 2 with 6N HCl. The volume was reduced to about one-third and 20 g of cation exchange resin (Strata) was added. The slurry was allowed to stand for 20 min and loaded onto a pad of cation exchange resin (Strata) (ca. 25 g). The pad was washed with H₂O (200 mL), MeOH (200 mL), and then NH₃ (3M in MeOH, 2×200 mL). The appropriate fractions was concentrated in vacuo and the residue (ca. 1.1 g) was dissolved in H₂O, frozen and lyophyllized. The title compound was obtained as a foam (1.02 g, 62%). ¹HNMR (400 MHz, DMSO-d₆) δ 8.00 (s, br, 1H), 7.68-7.71 (m, 1H), 7.01 (s, br, 1H), 6.88 (d, J=7.5 Hz, 1H), 5.75 (s, br, 1H), 3.54 (s, 1H), 2.04-2.06 (m, 1H), 0.95 (d, J=6.0 Hz, 3H), 0.91 (d, J=6.6 Hz, 3H). LCMS: Anal. Calcd. for C₁₀H₁₄N₂O₂: 194; found: 195 (M+H)⁺.

A mixture of L-valine (1.0 g, 8.54 mmol), 5-bromopyrimidine (4.03 g, 17.0 mmol), K₂CO₃ (2.40 g, 17.4 mmol) and CuI (179 mg, 0.94 mmol) in DMSO (10 mL) was heated at 100° C. for 12 h. The reaction mixture was cooled to RT, poured into H₂O (ca. 150 mL) and washed with EtOAc (×2). The organic layers were extracted with a small amount of H₂O and the combined aq phases were acidified to ca. pH 2 with 6N HCl. The volume was reduced to about one-third and 20 g of cation exchange resin (Strata) was added. The slurry was allowed to stand for 20 min and loaded onto a pad of cation exchange resin (Strata) (ca. 25 g). The pad was washed with H₂O (200 mL), MeOH (200 mL), and then NH₃ (3M in MeOH, 2×200 mL). The appropriate fractions was concentrated in vacuo and the residue (ca. 1.1 g) was dissolved in H₂O, frozen and lyophyllized. The title compound was obtained as a foam (1.02 g, 62%). ¹HNMR (400 MHz, CD₃OD) showed the mixture to contain valine and the purity could not be estimated. The material was used as is in subsequent reactions. LCMS: Anal. Calcd. for C₉H₁₃N₃O₂: 195; found: 196 (M+H)⁺.

Cap-90 was prepared according to the method described for the preparation of Cap-1. The crude material was used as is in subsequent steps. LCMS: Anal. Calcd. for C₁₁H₁₅NO₂: 193; found: 192 (M−H)⁻.

The following caps were prepared according to the method used for preparation of cap 51 unless noted otherwise:

Cap Structure Analytical Data Cap- 91

LCMS: Anal. Calcd. for C₁₁H₁₃NO₄: 223; found: 222 (M − H)⁻. Cap- 92

LCMS: Anal. Calcd. for C₁₁H₁₃NO₄: 223; found: 222 (M − H)⁻. Cap- 93

LCMS: Anal. Calcd. for C₁₀H₁₂N₂O₄: 224; found: 225 (M + H)⁺. Cap- 94

LCMS: Anal. Calcd. for C₈H₁₁N₃O₄: 213; found: 214 (M + H)⁺. Cap- 95

LCMS: Anal. Calcd. for C₁₃H₁₇NO₄: 251; found: 250 (M − H)⁻. Cap- 96

LCMS: Anal. Calcd. for C₁₂H₁₅NO₄: 237; found: 236 (M − H)⁻. Cap- 97

LCMS: Anal. Calcd. for C₉H₁₅NO₄: 201; found: 200 (M − H)⁻. Cap- 98

LCMS: Anal. Calcd. for C₉H₁₅NO₄: 201; found: 202 (M + H)⁺. Cap- 99

¹HNMR (400 MHz, CD₃OD) δ 3.88-3.94 (m, 1H), 3.60, 3.61 (s, 3H), 2.80 (m, 1H), 2.20 (m 1H), 1.82-1.94 (m, 3H), 1.45 - 1.71 (m, 2H). Cap- 99a

¹HNMR (400 MHz, CD₃OD) δ 3.88-3.94 (m, 1H), 3.60, 3.61 (s, 3H), 2.80 (m, 1H), 2.20 (m 1H), 1.82-1.94 (m, 3H), 1.45 - 1.71 (m, 2H). Cap- 100

LCMS: Anal. Calcd. for C₁₂H₁₄NO₄F: 255; found: 256 (M + H)⁺. Cap- 101

LCMS: Anal. Calcd. for C₁₁H₁₃NO₄: 223; found: 222 (M − H)⁻. Cap- 102

LCMS: Anal. Calcd. for C₁₁H₁₃NO₄: 223; found: 222 (M − H)⁻. Cap- 103

LCMS: Anal. Calcd. for C₁₀H₁₂N₂O₄: 224; found: 225 (M + H)⁺. Cap- 104

¹HNMR (400 MHz, CD₃OD) δ 3.60 (s, 3H), 3.50-3.53 (m, 1H), 2.66-2.69 and 2.44-2.49 (m, 1H), 1.91-2.01 (m, 2H), 1.62-1.74 (m, 4H), 1.51-1.62 (m, 2H). Cap- 105

¹HNMR (400 MHz, CD₃OD) δ 3.60 (s, 3H), 3.33-3.35 (m, 1H, partially obscured by solvent), 2.37-2.41 and 2.16-2.23 (m, 1H), 1.94-2.01 (m, 4H), 1.43-1.53 (m, 2H), 1.17-1.29 (m, 2H). Cap- 106

¹HNMR (400 MHz, CD₃OD) δ 3.16 (q, J = 7.3 Hz, 4H), 2.38-2.41 (m, 1H), 2.28-2.31 (m, 2H), 1.79-1.89 (m, 2H), 1.74 (app, ddd J = 3.5, 12.5, 15.9 Hz, 2H), 1.46 (app dt (J = 4.0, 12.9 Hz, 2H), 1.26 (t, J = 7.3 Hz, 6H) Cap- 107

LCMS: Anal. Calcd. for C₈H₁₀N₂O₄S: 230; found: 231 (M + H)⁺. Cap- 108

LCMS: Anal. Calcd. for C₁₅H₁₇N₃O₄: 303; found: 304 (M + H)⁺. Cap- 109

LCMS: Anal. Calcd. for C₁₀H₁₂N₂O₄: 224; found: 225 (M + H)⁺. Cap- 110

LCMS: Anal. Calcd. for C₁₀H₁₂N₂O₄: 224; found: 225 (M + H)⁺. Cap- 111

LCMS: Anal. Calcd. for C₁₂H₁₆NO₈P: 333; found: 334(M + H)⁺. Cap- 112

LCMS: Anal. Calcd. for C₁₃H₁₄N₂O₄: 262; found: 263 (M + H)⁺. Cap- 113

LCMS: Anal. Calcd. for C₁₈H₁₉NO₅: 329; found: 330 (M + H)⁺. Cap- 114

¹HNMR (400 MHz, CDCl₃) δ 4.82-4.84 (m, 1H), 4.00-4.05 (m, 2H), 3.77 (s, 3H), 2.56 (s, br, 2H) Cap- 115

¹HNMR (400 MHz, CDCl₃) δ 5.13 (s, br, 1H), 4.13 (s, br, 1H), 3.69 (s, 3H), 2.61 (d, J = 5.0 Hz, 2H), 1.28 (d, J = 9.1 Hz, 3H). Cap- 116

¹HNMR (400 MHz, CDCl₃) δ 5.10 (d, J = 8.6 Hz, 1H), 3.74-3.83 (m, 1H), 3.69 (s, 3H), 2.54-2.61 (m, 2H), 1.88 (sept, J = 7.0 Hz, 1H), 0.95 (d, J = 7.0 Hz, 6H).

Cap-117 to Cap-123

For the preparation of caps Cap-117 to Cap-123 the Boc amino acids were commercially available and were deprotected by treatment with 25% TFA in CH₂Cl₂. After complete reaction as judged by LCMS the solvents were removed in vacuo and the corresponding TFA salt of the amino acid was carbamoylated with methyl chloroformate according to the procedure for Cap-51.

Cap Structure Analytical Data Cap-117

LCMS: Anal. Calcd. for C₁₂H₁₅NO₄: 237; found: 238 (M + H)⁺. Cap-118

LCMS: Anal. Calcd. for C₁₀H₁₃NO₄S: 243; found: 244 (M + H)⁺. Cap-119

LCMS: Anal. Calcd. for C₁₀H₁₃NO₄S: 243; found: 244 (M + H)⁺. Cap-120

LCMS: Anal. Calcd. for C₁₀H₁₃NO₄S: 243; found: 244 (M + H)⁺. Cap-121

¹HNMR (400 MHz, CDCl₃) δ 4.06-4.16 (m, 1H), 3.63 (s, 3H), 3.43 (s, 1H), 2.82 and 2.66 (s, br, 1H), 1.86-2.10 (m, 3H), 1.64-1.76 (m, 2H), 1.44-1.53 (m, 1H). Cap-122

¹HNMR profile is similar to that of its enantiomer, Cap-121. Cap-123

LCMS: Anal. Calcd. for C₂₇H₂₆N₂O₆: 474; found: 475 (M + H)⁺.

Preparation of Cap-124. (4S,5R)-5-methyl-2-oxooxazolidine-4-carboxylic acid

The hydrochloride salt of L-threonine tert-butyl ester was carbamoylated according to the procedure for Cap-51. The crude reaction mixture was acidified with 1N HCl to pH ˜1 and the mixture was extracted with EtOAc (2×50 mL). The combined organic phases were concentrated in vacuo to give a colorless oil which solidified on standing. The aqueous layer was concentrated in vacuo and the resulting mixture of product and inorganic salts was triturated with EtOAc-CH₂Cl₂-MeOH (1:1:0.1) and then the organic phase concentrated in vacuo to give a colorless oil which was shown by LCMS to be the desired product. Both crops were combined to give 0.52 g of a solid. ¹HNMR (400 MHz, CD₃OD) δ 4.60 (m, 1H), 4.04 (d, J=5.0 Hz, 1H), 1.49 (d, J=6.3 Hz, 3H). LCMS: Anal. Calcd. for C₅H₇NO₄: 145; found: 146 (M+H)⁺.

Preparation of Cap-125. (S)-2-(tert-butoxycarbonylamino)-4-(dimethylamino)butanoic acid

To a suspension of Pd(OH)₂, (20%, 100 mg), aqueous formaldehyde (37% wt, 4 ml), acetic acid, (0.5 mL) in methanol (15 mL) was added (S)-4-amino-2-(tert-butoxycarbonylamino)butanoic acid (1 g, 4.48 mmol). The reaction was purged several times with hydrogen and was stirred overnight with an hydrogen balloon room temp. The reaction mixture was filtered through a pad of diatomaceous earth (Celite®), and the volatile component was removed in vacuo. The resulting crude material was used as is for the next step. LC/MS: Anal. Calcd. for C₁₁H₂₂N₂O₄: 246; found: 247 (M+H)⁺.

Preparation of 3-methyl-N-[(methyloxy)carbonyl]-L-histidine (Cap-126)

This procedure is a modification of that used to prepare Cap-51. To a suspension of 3-methyl-L-histidine (0.80 g, 4.70 mmol) in THF (10 mL) and H₂O (10 mL) at 0° C. was added NaHCO₃ (0.88 g, 10.5 mmol). The resulting mixture was treated with ClCO₂Me (0.40 mL, 5.20 mmol) and the mixture allowed to stir at 0° C. After stirring for ca. 2 h LCMS showed no starting material remaining. The reaction was acidified to pH 2 with 6 N HCl.

The solvents were removed in vacuo and the residue was suspended in 20 mL of 20% MeOH in CH₂Cl₂. The mixture was filtered and concentrated to give a light yellow foam (1.21 g,). LCMS and ¹H NMR showed the material to be a 9:1 mixture of the methyl ester and the desired product. This material was taken up in THF (10 mL) and H₂O (10 mL), cooled to 0° C. and LiOH (249.1 mg, 10.4 mmol) was added. After stirring ca. 1 h LCMS showed no ester remaining. Therefore the mixture was acidified with 6N HCl and the solvents removed in vacuo. LCMS and ¹H NMR confirm the absence of the ester. The title compound was obtained as its HCl salt contaminated with inorganic salts (1.91 g, >100%). The compound was used as is in subsequent steps without further purification. ¹HNMR (400 MHz, CD₃OD) δ 8.84, (s, 1H), 7.35 (s, 1H), 4.52 (dd, J=5.0, 9.1 Hz, 1H), 3.89 (s, 3H), 3.62 (s, 3H), 3.35 (dd, J=4.5, 15.6 Hz, 1H, partially obscured by solvent), 3.12 (dd, J=9.0, 15.6 Hz, 1H). LCMS: Anal. Calcd. for C₉H₁₃N₃O₄: 227.09; found: 228.09 (M+H)⁺.

Preparation of (S)-2-(methoxycarbonylamino)-3-(1-methyl-1H-imidazol-4-yl)propanoic acid (Cap-127)

Cap-127 was prepared according to the method for Cap-126 above starting from (S)-2-amino-3-(1-methyl-1H-imidazol-4-yl)propanoic acid (1.11 g, 6.56 mmol), NaHCO₃ (1.21 g, 14.4 mmol) and ClCO₂Me (0.56 mL, 7.28 mmol). The title compound was obtained as its HCl salt (1.79 g, >100%) contaminated with inorganic salts. LCMS and ¹H NMR showed the presence of ca. 5% of the methyl ester. The crude mixture was used as is without further purification. ¹HNMR (400 MHz, CD₃OD) δ 8.90 (s, 1H), 7.35 (s, 1H), 4.48 (dd, J=5.0, 8.6 Hz, 1H), 3.89 (s, 3H), 3.62 (s, 3H), 3.35 (m, 1H), 3.08 (m, 1H); LCMS: Anal. Calcd. for C₉H₁₃N₃O₄: 227.09; found: 228 (M+H)⁺.

Preparation of (S)-2-(methoxycarbonylamino)-3-(1H-1,2,3-triazol-4-yl)propanoic acid (Cap-128)

Step 1. Preparation of (S)-benzyl 2-(tert-butoxycarbonylamino)pent-4-ynoate (cj-27b).

To a solution of cj-27a (1.01 g, 4.74 mmol), DMAP (58 mg, 0.475 mmol) and iPr₂NEt (1.7 mL, 9.8 mmol) in CH₂Cl₂ (100 mL) at 0° C. was added Cbz-Cl (0.68 mL, 4.83 mmol). The solution was allowed to stir for 4 h at 0° C., washed (1N KHSO₄, brine), dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (TLC 6:1 hex:EtOAc) to give the title compound (1.30 g, 91%) as a colorless oil. ¹HNMR (400 MHz, CDCl₃) δ 7.35 (s, 5H), 5.35 (d, br, J=8.1 Hz, 1H), 5.23 (d, J=12.2 Hz, 1H), 5.17 (d, J=12.2 Hz, 1H), 4.48-4.53 (m, 1H), 2.68-2.81 (m, 2H), 2.00 (t, J=2.5 Hz, 1H), 1.44 (s, 9H). LCMS: Anal. Calcd. for C₁₇H₂₁NO₄: 303; found: 304 (M+H)⁺.

Step 2. Preparation of (S)-benzyl 3-(1-benzyl-1H-1,2,3-triazol-4-yl)-2-(tert-butoxycarbonylamino)propanoate (cj-28)

To a mixture of (S)-benzyl 2-(tert-butoxycarbonylamino)pent-4-ynoate (0.50 g, 1.65 mmol), sodium ascorbate (0.036 g, 0.18 mmol), CuSO₄-5H₂O (0.022 g, 0.09 mmol) and NaN₃ (0.13 g, 2.1 mmol) in DMF-H₂O (5 mL, 4:1) at rt was added BnBr (0.24 mL, 2.02 mmol) and the mixture was warmed to 65° C. After 5 h LCMS indicated low conversion. A further portion of NaN₃ (100 mg) was added and heating was continued for 12 h. The reaction was poured into EtOAc and H₂O and shaken. The layers were separated and the aqueous layer extracted 3× with EtOAc and the combined organic phases washed (H₂O ×3, brine), dried (Na₂SO₄), filtered, and concentrated. The residue was purified by flash (Biotage, 40+M 0-5% MeOH in CH₂Cl₂; TLC 3% MeOH in CH₂Cl₂) to afford a light yellow oil which solidified on standing (748.3 mg, 104%). The NMR was consistent with the desired product but suggests the presence of DMF. The material was used as is without further purification. ¹HNMR (400 MHz, DMSO-d₆) δ 7.84 (s, 1H), 7.27-7.32 (m, 10H), 5.54 (s, 2H), 5.07 (s, 2H), 4.25 (m, 1H), 3.16 (dd, J=1.0, 5.3 Hz, 1H), 3.06 (dd, J=5.3, 14.7 Hz), 2.96 (dd, J=9.1, 14.7 Hz, 1H), 1.31 (s, 9H).

LCMS: Anal. Calcd. for C₂₄H₂₈N₄O₄: 436; found: 437 (M+H)⁺.

Step 2. Preparation of (S)-benzyl 3-(1-benzyl-1H-1,2,3-triazol-4-yl)-2-(methoxycarbonylamino)propanoate (cj-29)

A solution of (S)-benzyl 3-(1-benzyl-1H-1,2,3-triazol-4-yl)-2-(tert-butoxycarbonylamino)propanoate (0.52 g, 1.15 mmol) in CH₂Cl₂ was added TFA (4 mL). The mixture was allowed to stir at room temperature for 2 h. The mixture was concentrated in vacuo to give a colorless oil which solidified on standing. This material was dissolved in THF-H₂O and cooled to 0° C. Solid NaHCO₃ (0.25 g, 3.00 mmol) was added followed by ClCO₂Me (0.25 mL, 3.25 mmol). After stirring for 1.5 h the mixture was acidified to pH-2 with 6N HCl and then poured into H₂O-EtOAc. The layers were separated and the aq phase extracted 2× with EtOAc. The combined org layers were washed (H₂O, brine), dried (Na₂SO₄), filtered, and concentrated in vacuo to give a colorless oil (505.8 mg, 111%, NMR suggested the presence of an unidentified impurity) which solidified while standing on the pump. The material was used as is without further purification. ¹HNMR (400 MHz, DMSO-d₆) δ 7.87 (s, 1H), 7.70 (d, J=8.1 Hz, 1H), 7.27-7.32 (m, 10H), 5.54 (s, 2H), 5.10 (d, J=12.7 Hz, 1H), 5.06 (d, J=12.7 Hz, 1H), 4.32-4.37 (m, 1H), 3.49 (s, 3H), 3.09 (dd, J=5.6, 14.7 Hz, 1H), 2.98 (dd, J=9.6, 14.7 Hz, 1H). LCMS: Anal. Calcd. for C₂₁H₂₂N₄O₄: 394; found: 395 (M+H)⁺.

Step 3. Preparation of (S)-2-(methoxycarbonylamino)-3-(1H-1,2,3-triazol-4-yl)propanoic acid (Cap-128)

(S)-benzyl 3-(1-benzyl-1H-1,2,3-triazol-4-yl)-2-(methoxycarbonylamino)propanoate (502 mg, 1.11 mmol) was hydrogenated in the presence of Pd—C (82 mg) in MeOH (5 mL) at atmospheric pressure for 12 h. The mixture was filtered through diatomaceous earth (Celite®) and concentrated in vacuo. (S)-2-(methoxycarbonylamino)-3-(1H-1,2,3-triazol-4-yl)propanoic acid was obtained as a colorless gum (266 mg, 111%) which was contaminated with ca. 10% of the methyl ester. The material was used as is without further purification. ¹HNMR (400 MHz, DMSO-d₆) δ 12.78 (s, br, 1H), 7.59 (s, 1H), 7.50 (d, J=8.0 Hz, 1H), 4.19-4.24 (m, 111), 3.49 (s, 3H), 3.12 (dd, J=4.8 Hz, 14.9 Hz, 1H), 2.96 (dd, J=9.9, 15.0 Hz, 1H). LCMS: Anal. Calcd. for C₇H₁₀N₄O₄: 214; found: 215 (M+H)⁺.

Preparation of (S)-2-(methoxycarbonylamino)-3-(1H-pyrazol-1-yl)propanoic acid (Cap-129)

Step 1. Preparation of (S)-2-(benzyloxycarbonylamino)-3-(1H-pyrazol-1-yl)propanoic acid (cj-31)

A suspension of (S)-benzyl 2-oxooxetan-3-ylcarbamate (0.67 g, 3.03 mmol), and pyrazole (0.22 g, 3.29 mmol) in CH₃CN (12 mL) was heated at 50° C. for 24 h. The mixture was cooled to rt overnight and the solid filtered to afford (S)-2-(benzyloxycarbonylamino)-3-(1H-pyrazol-1-yl)propanoic acid (330.1 mg). The filtrate was concentrated in vacuo and then triturated with a small amount of CH₃CN (ca. 4 mL) to afford a second crop (43.5 mg). Total yield 370.4 mg (44%). m.p. 165.5-168° C. lit m.p. 168.5-169.5 [Vederas et al. J. Am. Chem. Soc. 1985, 107, 7105]. ¹HNMR (400 MHz, CD₃OD) δ 7.51 (d, J=2.0, 1H), 7.48 (s, J=1.5 Hz, 1H), 7.24-7.34 (m, 5H), 6.23 m, 1H), 5.05 (d, 12.7 H, 1H), 5.03 (d, J=12.7 Hz, 1H), 4.59-4.66 (m, 2H), 4.42-4.49 (m, 1H). LCMS: Anal. Calcd. for C₁₄H₁₅N₃O₄: 289; found: 290 (M+H)⁺.

Step 2. Preparation of (S)-2-(methoxycarbonylamino)-3-(1H-pyrazol-1-yl)propanoic acid (Cap-129)

(S)-2-(benzyloxycarbonylamino)-3-(1H-pyrazol-1-yl)propanoic acid (0.20 g, 0.70 mmol) was hydrogenated in the presence of Pd—C (45 mg) in MeOH (5 mL) at atmospheric pressure for 2 h. The product appeared to be insoluble in MeOH, therefore the reaction mixture was diluted with 5 mL H₂O and a few drops of 6N HCl. The homogeneous solution was filtered through diatomaceous earth (Celite®), and the MeOH removed in vacuo. The remaining solution was frozen and lyophyllized to give a yellow foam (188.9 mg). This material was suspended in THF—H₂O (1:1, 10 mL) and then cooled to 0° C. To the cold mixture was added NaHCO₃ (146.0 mg, 1.74 mmol) carefully (evolution of CO₂). After gas evolution had ceased (ca. 15 min) ClCO₂Me (0.06 mL, 0.78 mmol) was added dropwise. The mixture was allowed to stir for 2 h and was acidified to pH ˜2 with 6N HCl and poured into EtOAc. The layers were separated and the aqueous phase extracted with EtOAC (×5). The combined organic layers were washed (brine), dried (Na₂SO₄), filtered, and concentrated to give the title compound as a colorless solid (117.8 mg, 79%). ¹HNMR (400 MHz, DMSO-d₆) δ 13.04 (s, 1H), 7.63 (d, J=2.6 Hz, 1H), 7.48 (d, J=8.1 Hz, 1H), 7.44 (d, J=1.5 Hz, 1H), 6.19 (app t, J=2.0 Hz, 1H), 4.47 (dd, J=3.0, 12.9 Hz, 1H), 4.29-4.41 (m, 2H), 3.48 (s, 3H). LCMS: Anal. Calcd. for C₈H₁₁N₃O₄: 213; found: 214 (M+H)⁺.

Cap-130. N-Acetyl —(R)-Phenylglycine

Cap-130 was prepared by acylation of commercially available (R)-phenylglycine analgous to the procedure given in: Calmes, M.; Daunis, J.; Jacquier, R.; Verducci, J. Tetrahedron, 1987, 43(10), 2285.

Step a: Dimethylcarbamoyl chloride (0.92 mL, 10 mmol) was added slowly to a solution of (S)-benzyl 2-amino-3-methylbutanoate hydrochloride (2.44 g; 10 mmol) and Hunig's base (3.67 mL, 21 mmol) in THF (50 mL). The resulting white suspension was stirred at room temperature overnight (16 hours) and concentrated under reduced pressure. The residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried (MgSO₄), filtered, and concentrated under reduced pressure. The resulting yellow oil was purified by flash chromatography, eluting with ethyl acetate:hexanes (1:1). Collected fractions were concentrated under vacuum providing 2.35 g (85%) of clear oil. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 0.84 (d, J=6.95 Hz, 3H) 0.89 (d, J=6.59 Hz, 3H) 1.98-2.15 (m, 1H) 2.80 (s, 6H) 5.01-5.09 (m, J=12.44 Hz, 1H) 5.13 (d, J=12.44 Hz, 1H) 6.22 (d, J=8.05 Hz, 1H) 7.26-7.42 (m, 5H). LC (Cond. 1): RT=1.76 min; MS: Anal. Calcd. for [M+H]⁺ C₁₆H₂₂N₂O₃: 279.17; found 279.03.

Step b: To a MeOH (50 mL) solution of the intermediate prepared above (2.35 g; 8.45 mmol) was added Pd/C (10%; 200 mg) and the resulting black suspension was flushed with N₂ (3×) and placed under 1 atm of H₂. The mixture was stirred at room temperature overnight and filtered though a microfiber filter to remove the catalyst. The resulting clear solution was then concentrated under reduced pressure to obtain 1.43 g (89%) of Cap-131 as a white foam, which was used without further purification. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.87 (d, J=4.27 Hz, 3H) 0.88 (d, J=3.97 Hz, 3H) 1.93-2.11 (m, 1H) 2.80 (s, 6H) 3.90 (dd, J=8.39, 6.87 Hz, 1H) 5.93 (d, J=8.54 Hz, 1H) 12.36 (s, 1H).). LC (Cond. 1): RT=0.33 min; MS: Anal. Calcd. for [M+H]⁺ C₈H₁₇N₂O₃: 1898.12; found 189.04.

Cap-132 was prepared from (S)-benzyl 2-aminopropanoate hydrochloride according to the method described for Cap-131. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.27 (d, J=7.32 Hz, 3H) 2.80 (s, 6H) 4.06 (qt, 1H) 6.36 (d, J=7.32 Hz, 1H) 12.27 (s, 1H). LC (Cond. 1): RT=0.15 min; MS: Anal. Calcd. for [M+H]⁺ C₆H₁₃N₂O₃: 161.09; found 161.00.

Cap-133 was prepared from (S)-tert-butyl 2-amino-3-methylbutanoate hydrochloride and 2-fluoroethyl chloroformate according to the method described for Cap-47. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.87 (t, J=6.71 Hz, 6H) 1.97-2.10 (m, 1H) 3.83 (dd, J=8.39, 5.95 Hz, 1H) 4.14-4.18 (m, 1H) 4.20-4.25 (m, 1H) 4.50-4.54 (m, 1H) 4.59-4.65 (m, 1H) 7.51 (d, J=8.54 Hz, 1H) 12.54 (s, 1H).

Cap-134 was prepared from (S)-diethyl alanine and methyl chloroformate according to the method described for Cap-51. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.72-0.89 (m, 6H) 1.15-1.38 (m, 4H) 1.54-1.66 (m, 1H) 3.46-3.63 (m, 3H) 4.09 (dd, J=8.85, 5.19 Hz, 1H) 7.24 (d, J=8.85 Hz, 1H) 12.55 (s, 1H). LC (Cond. 2): RT=0.66 min; LC/MS: Anal. Calcd. for [M+H]⁺ C₉H₁₈NO₄: 204.12; found 204.02.

A solution of D-2-amino-(4-fluorophenyl)acetic acid (338 mg, 2.00 mmol), 1N HCl in ether (2.0 mL, 2.0 mmol) and formalin (37%, 1 mL) in methanol (5 mL) was subjected to balloon hydrogenation over 10% palladium on carbon (60 mg) for 16 h at 25° C. The mixture was then filtered through Celite to afford Cap-135 as a white foam (316 mg, 80%). ¹H NMR (300 MHz, MeOH-d₄) □ 7.59 (dd, J=8.80, 5.10 Hz, 2H), 7.29 (t, J=8.6 Hz, 2H), 5.17 (s, 1H), 3.05 (v br s, 3H), 2.63 (v br s, 3H); R_(t)=0.19 min (Cond.—MS-W5); 95% homogeneity index; LRMS: Anal. Calc. for [M+H]⁺ C₁₀H₁₃FNO₂: 198.09; found: 198.10.

To a cold (−50° C.) suspension of 1-benzyl-1H-imidazole (1.58 g, 10.0 mmol) in anhydrous diethyl ether (50 mL) under nitrogen was added n-butyl lithium (2.5 M in hexanes, 4.0 mL, 10.0 mmol) dropwise. After being stirred for 20 min at −50° C., dry carbon dioxide (passed through Drierite) was bubbled into the reaction mixture for 10 min before it was allowed to warm up to 25° C. The heavy precipitate which formed on addition of carbon dioxide to the reaction mixture was filtered to yield a hygroscopic, white solid which was taken up in water (7 mL), acidified to pH=3, cooled, and induced to crystallize with scratching. Filtration of the precipitate gave a white solid which was suspended in methanol, treated with 1N HCl/diethyl ether (4 mL) and concentrated in vacuo. Lyophilization of the residue from water (5 mL) afforded Cap-136 as a white solid (817 mg, 40%). ¹H NMR (300 MHz, DMSO-d₆) δ 7.94 (d, J=1.5 Hz, 1H), 7.71 (d, J=1.5 Hz, 1H), 7.50-7.31 (m, 5H), 5.77 (s, 2H); R_(t)=0.51 min (Cond.—MS-W5); 95% homogeneity index; LRMS: Anal. Calc. for [M+H]⁺ C₁₁H₁₂N₂O₂: 203.08; found: 203.11.

A suspension of 1-chloro-3-cyanoisoquinoline (prepared according to the procedure in WO 2003/099274) (188 mg, 1.00 mmol), cesium fluoride (303.8 mg, 2.00 mmol), bis(tri-tert-butylphosphine)palladium dichloride (10 mg, 0.02 mmol) and 2-(tributylstannyl)furan (378 μL, 1.20 mmol) in anhydrous dioxane (10 mL) under nitrogen was heated at 80° C. for 16 h before it was cooled to 25° C. and treated with saturated, aqueous potassium fluoride solution with vigorous stirring for 1 h. The mixture was partitioned between ethyl acetate and water and the organic phase was separated, washed with brine, dried with Na₂SO₄, filtered and concentrated. Purification of the residue on silica gel eluting with 0% to 30% ethyl acetate/hexanes afforded Cap-137, step a as a white solid which was used as is (230 mg, 105%). R_(t)=1.95 min (Cond.—MS-W2); 90% homogeneity index; LRMS: Anal. Calc. for [M+H]⁺ C₁₄H₈N₂O: 221.07; found: 221.12.

To a suspension of Cap 137, step a, (110 mg, 0.50 mmol) and sodium periodate (438 mg, 2.05 mmol) in carbon tetrachloride (1 mL), acetonitrile (1 mL) and water (1.5 mL) was added ruthenium trichloride hydrate (2 mg, 0.011 mmol) in one portion. The mixture was stirred at 25° C. for 2 h and then partitioned between dichloromethane and water. The aqueous layer was separated, extracted twice more with dichloromethane and the combined dichloromethane extracts were dried with Na₂SO₄, filtered and concentrated. Trituration of the residue with hexanes afforded Cap-137 (55 mg, 55%) as a grayish-colored solid. R_(t)=1.10 min (Cond.—MS-W2); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₁H₈N₂O₂: 200.08; found: 200.08.

Caps 138 to 158

Synthetic Strategy. Method A.

To a stirred suspension of 5-hydroxyisoquinoline (prepared according to the procedure in WO 2003/099274) (2.0 g, 13.8 mmol) and triphenylphosphine (4.3 g, 16.5 mmol) in dry tetrahydrofuran (20 mL) was added dry methanol (0.8 mL) and diethyl azodicarboxylate (3.0 mL, 16.50 mmol) portionwise. The mixture was stirred at room temperature for 20 h before it was diluted with ethyl acetate and washed with brine, dried with Na₂SO₄, filtered and concentrated. The residue was preabsorbed onto silica gel and purified, eluting with 40% ethyl acetate/hexanes to afford Cap-138, step a as a light yellow solid (1.00 g, 45%). ¹H NMR (CDCl₃, 500 MHz) δ 9.19 (s, 1H), 8.51 (d, J=6.0 Hz, 1H), 7.99 (d, J=6.0 Hz, 1H), 7.52-7.50 (m, 2H), 7.00-6.99 (m, 1H), 4.01 (s, 3H); R_(t)=0.66 min (Cond. D2); 95% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₀H₁₀NO: 160.08; found 160.1.

To a stirred solution of Cap 138, step a (2.34 g, 14.7 mmol) in anhydrous dichloromethane (50 mL) at room temperature was added meta-chloroperbenzoic acid (77%, 3.42 g, 19.8 mmol) in one portion. After being stirred for 20 h, powdered potassium carbonate (2.0 g) was added and the mixture was stirred for 1 h at room temperature before it was filtered and concentrated to afford Cap-138, step b as a pale, yellow solid which was sufficiently pure to carry forward (2.15 g, 83.3%). ¹H NMR (CDCl₃, 400 MHz) δ 8.73 (d, J=1.5 Hz, 1H), 8.11 (dd, J=7.3, 1.7 Hz, 1H), 8.04 (d, J=7.1 Hz, 1H), 7.52 (t, J=8.1 Hz, 1H), 7.28 (d, J=8.3 Hz, 1H), 6.91 (d, J=7.8 Hz, 1H), 4.00 (s, 3H); R_(t)=0.92 min, (Cond.—D1); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₀H₁₀NO₂: 176.07; found: 176.0.

To a stirred solution of Cap 138, step b (0.70 g, 4.00 mmol) and triethylamine (1.1 mL, 8.00 mmol) in dry acetonitrile (20 mL) at room temperature under nitrogen was added trimethylsilylcyanide (1.60 mL, 12.00 mmol). The mixture was heated to 75° C. for 20 h before it was cooled to room temperature, diluted with ethyl acetate and washed with saturated sodium bicarbonate solution and brine prior to drying with Na₂SO₄ and solvent concentration. The residue was flash chromatographed on silica gel eluting with 5% ethyl acetate/hexanes to 25% ethyl acetate/hexanes to afford Cap-138, step c (498.7 mg) as a white, crystalline solid along with 223 mg of additional Cap-138, step c recovered from the filtrate; ¹H NMR (CDCl₃, 500 MHz) δ 8.63 (d, J=5.5 Hz, 1H), 8.26 (d, J=5.5 Hz, 1H), 7.88 (d, J=8.5 Hz, 1H), 7.69 (t, J=8.0 Hz, 1H), 7.08 (d, J=7.5 Hz, 1H), 4.04 (s, 3H); R_(t)=1.75 min, (Cond.—D1); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₁H₉N₂O: 185.07; found: 185.10.

Cap-138, step c (0.45 g, 2.44 mmol) was treated with 5N sodium hydroxide solution (10 mL) and the resulting suspension was heated to 85° C. for 4 h, cooled to 25° C., diluted with dichloromethane and acidified with 1N hydrochloric acid. The organic phase was separated, washed with brine, dried, concentrated to ¼ volume and filtered to afford Cap-138 as a yellow solid (0.44 g, 88.9%). ¹H NMR (DMSO-d₆, 400 MHz) δ 13.6 (br s, 1H), 8.56 (d, J=6.0 Hz, 1H), 8.16 (d, J=6.0 Hz, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.71-7.67 (m, 1H), 7.30 (d, J=8.0 Hz, 1H), 4.02 (s, 3H); R_(t)=0.70 min (Cond.—D1); 95% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₁H₁₀NO₃: 204.07; found: 204.05.

Synthetic Strategy. Method B (derived from Tetrahedron Letters, 2001, 42, 6707).

To a thick-walled, screw-top vial containing an argon-degassed suspension of 1-chloro-6-methoxyisoquinoline (prepared according to the procedure in WO 2003/099274) (1.2 g, 6.2 mmol), potassium cyanide (0.40 g, 6.2 mmol), 1,5-bis(diphenylphosphino)pentane (0.27 g, 0.62 mmol) and palladium (II) acetate (70 mg, 0.31 mmol) in anhydrous toluene (6 mL) was added N,N,N′,N′-tetramethylethylenediamine (0.29 mL, 2.48 mmol). The test tube was sealed and heated to 150° C. for 22 h, and then allowed to cool to 25° C. The reaction was diluted with ethyl acetate, washed with water and brine, dried with Na₂SO₄, filtered and concentrated. The residue was purified on silica gel eluting with 5% ethyl acetate/hexanes to 25% ethyl acetate/hexanes to afford Cap-139, step a as a white solid (669.7 mg). ¹H NMR (CDCl₃, 500 MHz) δ 8.54 (d, J=6.0 Hz, 1H), 8.22 (d, J=9.0 Hz, 1H), 7.76 (d, J=5.5 Hz, 1H), 7.41-7.39 (m, 1H), 7.13 (d, J=2.0 Hz, 1H), 3.98 (s, 3H); R_(t)=1.66 min (Cond.—D1); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₁H₉N₂O: 185.07; found: 185.2.

Cap-139 was prepared from the basic hydrolysis of Cap-139, step a with 5N NaOH according to the procedure described for Cap 138. ¹H NMR (400 MHz, DMSO-d₆) δ 13.63 (v br s, 1H), 8.60 (d, J=9.3 Hz, 1H), 8.45 (d, J=5.6 Hz, 1H), 7.95 (d, J=5.9 Hz, 1H), 7.49 (d, J=2.2 Hz, 1H), 7.44 (dd, J=9.3, 2.5 Hz, 1H), 3.95 (s, 3H); R_(t)=0.64 min (Cond.—D1); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₁H₁₀NO₃: 204.07; found: 204.05.

To a vigorously-stirred mixture of 1,3-dichloro-5-ethoxyisoquinoline (prepared according to the procedure in WO 2005/051410) (482 mg, 2.00 mmol), palladium (II) acetate (9 mg, 0.04 mmol), sodium carbonate (223 mg, 2.10 mmol) and 1,5-bis(diphenylphosphino)pentane (35 mg, 0.08 mmol) in dry dimethylacetamide (2 mL) at 25° C. under nitrogen was added N,N,N′,N′-tetramethylethylenediamine (60 mL, 0.40 mmol). After 10 min, the mixture was heated to 150° C. A stock solution of acetone cyanohydrin (prepared from 457 μL of acetone cyanohydrin in 4.34 mL DMA) was added in 1 mL portions over 18 h using a syringe pump. The mixture was then partitioned between ethyl acetate and water and the organic layer was separated, washed with brine, dried with Na₂SO₄, filtered and concentrated. The residue was purified on silica gel eluting with 10% ethyl acetate/hexanes to 40% ethyl acetate/hexanes to afford Cap-101, step a as a yellow solid (160 mg, 34%). R_(t)=2.46 min (Cond.—MS-W2); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₂H₉ClN₂O: 233.05; found: 233.08.

Cap-140 was prepared by the acid hydrolysis of Cap-140, step a with 12N HCl as described in the procedure for the preparation of Cap 141. R_(t)=2.24 min (Cond.—MS-W2); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₂H₃₁ClNO₃: 252.04; found: 252.02.

Cap-141, step a was prepared from 1-bromo-3-fluoroisoquinoline (prepared from 3-amino-1-bromoisoquinoline using the procedure outlined in J. Med. Chem. 1970, 13, 613) as described in the procedure for the preparation of Cap-140, step a vide infra. ¹H NMR (500 MHz, CDCl₃) δ 8.35 (d, J=8.5 Hz, 1H), 7.93 (d, J=8.5 Hz, 1H), 7.83 (t, J=7.63 Hz, 1H), 7.77-7.73 (m, 1H), 7.55 (s, 1H); R_(t)=1.60 min (Cond.—D1); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₀H₆FN₂: 173.05; found: 172.99.

Cap-141, step a (83 mg, 0.48 mmol) was treated with 12N HCl (3 mL) and the resulting slurry was heated to 80° C. for 16 h before it was cooled to room temperature and diluted with water (3 mL). The mixture was stirred for 10 min, and then filtered to afford Cap-141 as an off-white solid (44.1 mg, 47.8%). The filtrate was diluted with dichloromethane and washed with brine, dried and concentrated to afford additional Cap-141 which was sufficiently pure to be carried forward directly (29.30 mg, 31.8%). ¹H NMR (DMSO-d₆, 500 MHz) δ 14.0 (br s, 1H), 8.59-8.57 (m, 1H), 8.10 (d, J=8.5 Hz, 1H), 7.88-7.85 (m, 2H), 7.74-7.71 (m, 1H); R_(t)=1.33 min (Cond.—D1); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₀H₇FNO₂: 192.05; found: 191.97.

Cap-142 step a was prepared from 4-bromoisoquinoline N-oxide as described in the two-step procedure for the preparation of Cap-138 steps b and c. R_(t)=1.45 min (Cond.—MS-W1); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₀H₆BrN₂: 232.97; found: 233.00.

To an argon-degassed suspension of Cap-142, step a (116 mg, 0.50 mmol), potassium phosphate tribasic (170 mg, 0.80 mmol), palladium (II) acetate (3.4 mg, 0.015 mmol) and 2-(dicyclohexylphosphino)biphenyl (11 mg, 0.03 mmol) in anhydrous toluene (1 mL) was added morpholine (61 μL, 0.70 mmol). The mixture was heated to 100° C. for 16 h, cooled to 25° C. and filtered through diatomaceous earth)(Celite®). Purification of the residue on silica gel, eluting with 10% to 70% ethyl acetate/hexanes afforded Cap-142, step b (38 mg, 32%) as a yellow solid, which was carried forward directly. R_(t)=1.26 min (Cond.—MS-W1); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₄H₁₄N₃O: 240.11; found: 240.13.

Cap-142 was prepared from Cap-142, step b with 5N sodium hydroxide as described in the procedure for Cap 138. R_(t)=0.72 min (Cond.—MS-W1); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₄H₁₅N₂O₃: 259.11; found: 259.08.

To a stirred solution of 3-amino-1-bromoisoquinoline (444 mg, 2.00 mmol) in anhydrous dimethylformamide (10 mL) was added sodium hydride (60%, unwashed, 96 mg, 2.4 mmol) in one portion. The mixture was stirred at 25° C. for 5 min before 2-bromoethyl ether (90%, 250 □L, 2.00 mmol) was added. The mixture was stirred at 25° C. for 5 h and at 75° C. for 72 h before it was cooled to 25° C., quenched with saturated ammonium chloride solution and diluted with ethyl acetate. The organic layer was separated, washed with water and brine, dried with Na₂SO₄, filtered and concentrated. Purification of the residue on silica gel eluting with 0% to 70% ethyl acetate/hexanes afforded Cap-143, step a as a yellow solid (180 mg, 31%). R_(t)=1.75 min (Cond.—MS-W1); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₃H₁₄BrN₂O: 293.03; found: 293.04.

To a cold (−60° C.) solution of Cap-143, step a (154 mg, 0.527 mmol) in anhydrous tetrahydrofuran (5 mL) was added a solution of n-butyllithium in hexanes (2.5 M, 0.25 mL, 0.633 mmol). After 10 min, dry carbon dioxide was bubbled into the reaction mixture for 10 min before it was quenched with 1N HCl and allowed to warm to 25° C. The mixture was then extracted with dichloromethane (3×30 mL) and the combined organic extracts were concentrated in vacuo. The residue was purified by a reverse phase HPLC (MeOH/water/TFA) to afford Cap-143 (16 mg, 12%). R_(t)=1.10 min (Cond.—MS-W1); 90% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₄H₁₅N₂O₃: 259.11; found: 259.08.

1,3-Dichloroisoquinoline (2.75 g, 13.89 mmol) was added in small portions to a cold (0° C.) solution of fuming nitric acid (10 mL) and concentrated sulfuric acid (10 mL). The mixture was allowed to stir at 0° C. for 0.5 h before it was gradually warmed to 25° C. where it stirred for 16 h. The mixture was then poured into a beaker containing chopped ice and water and the resulting suspension was stirred for 1 h at 0° C. before it was filtered to afford Cap-144, step a (2.73 g, 81%) as a yellow solid which was used as is. R_(t)=2.01 min. (Cond.—D1); 95% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₉H₅Cl₂N₂O₂: 242.97; found: 242.92.

Cap-144, step a (0.30 g, 1.23 mmol) was taken up in methanol (60 mL) and treated with platinum oxide (30 mg). The suspension was subjected to Parr hydrogenation (7 psi H₂) for 1.5 h before formalin (5 mL) and additional platinum oxide (30 mg) were added. The suspension was resubjected to Parr hydrogenation at 45 psi H₂ for 13 h before it was suction-filtered through diatomaceous earth (Celite®) and concentrated down to ¼ volume. Suction-filtration of the ensuing precipitate afforded the title compound as a yellow solid which was flash chromatographed on silica gel eluting with 5% ethyl acetate in hexanes to 25% ethyl acetate in hexanes to afford Cap-144, step b (231 mg, 78%) as a pale yellow solid. R_(t)=2.36 min (Cond.—D1); 95% homogeneity index; ¹H NMR (400 MHz, CDCl₃) δ 8.02 (s, 1H), 7.95 (d, J=8.6 Hz, 1H), 7.57-7.53 (m, 1H), 7.30 (d, J=7.3 Hz, 111), 2.88 (s, 6H); LCMS: Anal. Calc. for [M+H]⁺ C₁₁H₁₁Cl₂N₂: 241.03; found: 241.02. HRMS: Anal. Calc. for [M+H]⁺ C₁₁H₁₁Cl₂N₂: 241.0299; found: 241.0296.

Cap-144, step c was prepared from Cap-144, step b according to the procedure described for the preparation of Cap-139, step a. R_(t)=2.19 min. (Cond.—D1); 95% homogeneity index; LCMS: Anal. Calc. for [M+H]⁺ C₁₂H₁₁ClN₃: 232.06; found: 232.03. HRMS: Anal. Calc. for [M+H]⁺ C₁₂H₁₁ClN₃: 232.0642; found: 232.0631.

Cap-144 was prepared according to the procedure described for Cap-141. R_(t)=2.36 min (Cond.—D1); 90%; LCMS: Anal. Calc. for [M+H]⁺ C₁₂H₁₂ClN₂O₂: 238.01; found: 238.09.

Caps-145 to 162

Caps-145 to 162 were prepared from the appropriate 1-chloroisoquinolines according to the procedure described for the preparation of Cap-138 (Method A) or Cap 139 (Method B) unless noted otherwise as outlined below.

R_(t) (LC- Cond.); % homogeneity index; MS Cap # Cap Method Hydrolysis data Cap-145

B 12N HCl 1.14 min (Cond.-MS- W1); 90%; LCMS: Anal. Calc. for [M + H]⁺ C₁₀H₇ClNO₂: 208.02; found: 208.00. Cap-146

A 5N NaOH 1.40 min (Cond.-D1); 95% ; LCMS: Anal. Calc. for [M + H]⁺ C₁₁H₁₀NO₃: 204.07; found: 204.06. Cap-147

B 5N NaOH 0.87 min (Cond.-D1); 95%; LCMS: Anal. Calc. for [M + H]⁺ C₁₁H₁₀NO₃: 204.07; found: 204.05. Cap-148

A 5N NaOH 0.70 min (Cond.-D1); 95%; LCMS: Anal. Calc. for [M + H]⁺ C₁₁H₁₀NO₃: 204.07; found: 204.05. Cap-149

A 5N NaOH 0.70 min (Cond.-D1); 95%; LCMS: Anal. Calc. for [M + H]⁺ C₁₁H₁₀NO₃: 204.07; found: 204.05. Cap-150

A 12N HCl 0.26 min (Cond.-D1); 95%; LCMS: Anal. Calc. for [M + H]⁺ C₁₁H₁₀NO₃: 204.07; found: 204.04 . Cap-151

B 12N HCl 1.78 min (Cond.-D1); 90%; LCMS: Anal. Calc. for [M + H]⁺ C₁₁H₉ClNO₃: 238.03; found: 238.09. Cap-152

B 12N HCl 1.65 min (Cond.-D1); 95%; LCMS: Anal. Calc. for [M + H]⁺ C₁₁H₉ClNO₃: 238.00; found: 238.09. Cap-153

A 6N HCl 1.18 min (Cond.-MS- W1); 95%; LCMS: Anal. Calc. for [M + H]⁺ C₁₀H₇BrNO₂: 251.97; found: 251.95. Cap-154

B 5N NaOH 0.28 min (Cond.-MS- W1); 90%; LCMS: Anal. Calc. for [M + H]⁺ C₁₀H₇FNO₂: 192.05; found: 192.03. Cap-155

B 5N NaOH 0.59 min (Cond.-MS- W1); 90%; LCMS: Anal. Calc. for [M + H]⁺ C₁₀H₇ClNO₂: 208.02; found: 208.00. Cap-156

B 5N NaOH 0.60 min (Cond.-MS- W1); 90%; LCMS: Anal. Calc. for [M + H]⁺ C₁₀H₇ClNO₂: 208.02; found: 208.03. Cap-157

B 12N HCl 1.49 min (Cond.-D1); 95%; LCMS: Anal. Calc. for [M + H]⁺ C₁₀H₁₇ClNO₂: 208.02; found: 208.00. Cap-158

B 5N NaOH 0.69 min (Cond.-MS- W1); 90%; LCMS: Anal. Calc. for [M + H]⁺ C₁₀H₇ClNO₂: 208.02; found: 208.01. Cap-159

B 5N NaOH 0.41 min (Cond.-MS- W1); 90%; LCMS: Anal. Calc. for [M + H]⁺ C₁₀H₇FNO₂: 192.05; found: 192.03. Cap-160

B 5N NaOH 0.30 min (Cond.-MS- W1); 90%; LCMS: Anal. Calc. for [M + H]⁺ C₁₀H₇FNO₂: 192.05; found: 192.03. Cap-161

— — 0.70 min (Cond. D1); 95%; LCMS: Anal. Calc. for [M + H]⁺ C₁₂H₁₃N₂O₂: 217.10; found: 217.06. Cap-162

— — 0.65 min (Cond.-M3); 95%; LCMS: Anal. Calc. for [M + H]⁺ C₁₁H₁₀NO₃: 204.07; found: 203.94.

To a solution of 2-ketobutyric acid (1.0 g, 9.8 mmol) in diethylether (25 ml) was added phenylmagnesium bromide (22 ml, 1M in THF) dropwise. The reaction was stirred at ˜25° C. under nitrogen for 17.5 h. The reaction was acidified with 1N HCl and the product was extracted with ethyl acetate (3×100 ml). The combined organic layer was washed with water followed by brine and dried over MgSO₄. After concentration in vacuo, a white solid was obtained. The solid was recrystallized from hexanes/ethyl acetate to afford Cap-163 as white needles (883.5 mg). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 500 MHz): 12.71 (br s, 1 H), 7.54-7.52 (m, 2H), 7.34-7.31 (m, 2H), 7.26-7.23 (m, 1H), 5.52-5.39 (br s, 1H), 2.11 (m, 1H), 1.88 (m, 1H), 0.79 (app t, J=7.4 Hz, 3H).

A mixture of 2-amino-2-phenylbutyric acid (1.5 g, 8.4 mmol), formaldehyde (14 mL, 37% in water), 1N HCl (10 mL) and 10% Pd/C (0.5 mg) in MeOH (40 mL) was exposed to H₂ at 50 psi in a Parr bottle for 42 h. The reaction was filtered over Celite and concentrated in vacuo, the residue was taken up in MeOH (36 mL) and the product was purified with a reverse phase HPLC (MeOH/H₂O/TFA) to afford the TFA salt of Cap-164 as a white solid (1.7 g). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 500 MHz) 7.54-7.47 (m, 5H), 2.63 (m, 1H), 2.55 (s, 6H), 2.31 (m, 1H), 0.95 (app t, J=7.3 Hz, 3H).

To a mixture of 2-amino-2-indanecarboxylic acid (258.6 mg, 1.46 mmol) and formic acid (0.6 ml, 15.9 mmol) in 1,2-dichloroethane (7 ml) was added formaldehyde (0.6 ml, 37% in water). The mixture was stirred at ˜25° C. for 15 min then heated at 70° C. for 8 h. The volatile component was removed in vacuo, and the residue was dissolved in DMF (14 mL) and purified by a reverse phase HPLC (MeOH/H₂O/TFA) to afford the TFA salt of Cap-165 as a viscous oil (120.2 mg). ¹H NMR (DMSO-d₆, δ=2.5 ppm, 500 MHz): 7.29-7.21 (m, 4 H), 3.61 (d, J=17.4 Hz, 2H), 3.50 (d, J=17.4 Hz, 2H), 2.75 (s, 6H). LC/MS: Anal. Calcd. for [M+H]⁺ C₁₂H₁₆NO₂: 206.12; found: 206.07.

Caps-166a and -166b were prepared from (1S,4S)-(+)-2-methyl-2,5-diazabicyclo[2.2.1]heptane (2HBr) according to the method described for the synthesis of Cap-7a and Cap-7b, with the exception that the benzyl ester intermediate was separated using a semi-prep Chrialcel OJ column, 20×250 mm, 10 μm eluting with 85:15 heptane/ethanol mixture at 10 mL/min elution rate for 25 min. Cap-166b: ¹H NMR (DMSO-d₆, 68=2.5 ppm, 500 MHz): 7.45 (d, J=7.3 Hz, 2H), 7.27-7.19 (m, 3H), 4.09 (s, 1H), 3.34 (app br s, 1H), 3.16 (app br s, 1H), 2.83 (d, J=10.1 Hz, 1H), 2.71 (m, 2H), 2.46 (m, 1H), 2.27 (s, 3H), 1.77 (d, J=9.8 Hz, 1H), 1.63 (d, J=9.8 Hz, 1H). LC/MS: Anal. Calcd. for [M+H]⁺ C₁₄H₁₉N₂O₂: 247.14; found: 247.11.

A solution of racemic Boc-1,3-dihydro-2H-isoindole carboxylic acid (1.0 g, 3.8 mmol) in 20% TFA/CH₂Cl₂ was stirred at ˜25° C. for 4 h. All the volatile component was removed in vacuo. A mixture of the resultant crude material, formaldehyde (15 mL, 37% in water), 1N HCl (10 mL) and 10% Pd/C (10 mg) in MeOH was exposed to H₂ (40 PSI) in a Parr bottle for 23 h. The reaction mixture was filtered over Celite and concentrated in vacuo to afford Cap-167 as a yellow foam (873.5 mg). NMR (DMSO-d₆, δ =2.5 ppm, 500 MHz) 7.59-7.38 (m, 4H), 5.59 (s, 1H), 4.84 (d, J=14 Hz, 1H), 4.50 (d, J=14.1 Hz, 1H), 3.07 (s, 3H). LC/MS: Anal. Calcd. for [M+H]⁺ C₃₀H₁₂NO₂: 178.09; found: 178.65.

Racemic Cap-168 was prepared from racemic Boc-aminoindane-1-carboxylic acid according to the procedure described for the preparation of Cap-167. The crude material was employed as such.

A mixture of 2-amino-2-phenylpropanoic acid hydrochloride (5.0 g, 2.5 mmol), formaldehyde (15 ml, 37% in water), 1N HCl (15 ml), and 10% Pd/C (1.32 g) in MeOH (60 mL) was placed in a Parr bottle and shaken under hydrogen (55 PSI) for 4 days. The reaction mixture was filtered over Celite and concentrated in vacuo. The residue was taken up in MeOH and purified by reverse phase prep-HPLC (MeOH/water/TFA) to afford the TFA salt of Cap-269 as a viscous semi-solid (2.1 g). ¹H NMR (CDCl₃, δ=7.26 ppm, 500 MHz): 7.58-7.52 (m, 2 H), 7.39-7.33 (m, 3H), 2.86 (br s, 3H), 2.47 (br s, 3H), 1.93 (s, 3H). LC/MS: Anal. Calcd. for [M+H]⁺ C₁₁H₁₆NO₂: 194.12; found: 194.12.

To (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (505 mg; 3.18 mmol; obtained from Astatech) in water (15 mL) was added sodium carbonate (673 mg; 6.35 mmol) and the resultant mixture was cooled to 0° C. at which time methyl chloroformate (0.26 mL; 3.33 mmol) was added dropwise over 5 minutes. The reaction was allowed to stir for 18 hours slowly coming to ambient temperature as the ice bath melted. The reaction mixture was then partitioned between 1N HCl and ethyl acetate. The organic layer was removed and the aqueous layer was further extracted with 2 additional portions ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated in vacuo to a colorless residue. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 12.65 (1 H, br. s.), 7.44 (1 H, d, J=8.24 Hz), 3.77-3.95 (3 H, m), 3.54 (3 H, s), 3.11-3.26 (2 H, m), 1.82-1.95 (1 H, m), 1.41-1.55 (2 H, m), 1.21-1.39 (2 H, m); LC/MS: Anal. Calcd. for [M+H]⁺ C₉H₁₆NO₅: 218.1; found 218.1.

A solution of methyl 2-(benzyloxycarbonylamino)-2-(oxetan-3-ylidene)acetate (200 mg, 0.721 mmol; Il Farmaco (2001), 56, 609-613) in ethyl acetate (7 ml) and CH₂Cl₂ (4.00 ml) was degassed by bubbling nitrogen for 10 min. Dimethyl dicarbonate (0.116 ml, 1.082 mmol) and Pd/C (20 mg, 0.019 mmol) were then added, the reaction mixture was fitted with a hydrogen balloon and allowed to stir at ambient temperature overnight at which time TLC (95:5 CH₂Cl₂/MeOH: visualized with stain made from 1 g Ce(NH₄)₂SO₄, 6 g ammonium molybdate, 6 mL sulfuric acid, and 100 mL water) indicated complete conversion. The reaction was filtered through celite and concentrated. The residue was purified via Biotage® (load with dichloromethane on 25 samplet; elute on 25S column with dichloromethane for 3CV then 0 to 5% MeOH/dichloromethane over 250 mL then hold at 5% MeOH/dichloromethane for 250 mL; 9 mL fractions). Collected fractions containing desired material and concentrated to 120 mg (81%) of methyl 2-(methoxycarbonylamino)-2-(oxetan-3-yl)acetate as a colorless oil. ¹H NMR (500 MHz, CHLOROFORM-D) δ ppm 3.29-3.40 (m, J=6.71 Hz, 1 H) 3.70 (s, 3 H) 3.74 (s, 3 H) 4.55 (t, J=6.41 Hz, 1 H) 4.58-4.68 (m, 2 H) 4.67-4.78 (m, 2 H) 5.31 (br s, 1 H). LC/MS: Anal. Calcd. for [M+H]⁺ C₈H₁₄NO₅: 204.2; found 204.0.

To methyl 2-(methoxycarbonylamino)-2-(oxetan-3-yl)acetate (50 mg, 0.246 mmol) in THF (2 mL) and water (0.5 mL) was added lithium hydroxide monohydrate (10.33 mg, 0.246 mmol). The resultant solution was allowed to stir overnite at ambient temperature. TLC (1:1 EA/Hex; Hanessian stain [1 g Ce(NH₄)₂SO₄, 6 g ammonium molybdate, 6 mL sulfuric acid, and 100 mL water]) indicated ˜10% starting material remaining. Added an additional 3 mg LiOH and allowed to stir overnight at which time TLC showed no starting material remaining. Concentrated in vacuo and placed on high vac overnite providing 55 mg lithium 2-(methoxycarbonylamino)-2-(oxetan-3-yl)acetate as a colorless solid. ¹H NMR (500 MHz, MeOD) δ ppm 3.39-3.47 (m, 1 H) 3.67 (s, 3 H) 4.28 (d, J=7.93 Hz, 1 H) 4.64 (t, J=6.26 Hz, 1 H) 4.68 (t, J=7.02 Hz, 1 H) 4.73 (d, J=7.63 Hz, 2 H).

The following diazotization step was adapted from Barton, A.; Breukelman, S. P.; Kaye, P. T.; Meakins, G. D.; Morgan, D. J. J. C. S. Perkin Trans I 1982, 159-164: A solution of NaNO₂ (166 mg, 2.4 mmol) in water (0.6 mL) was added slowly to a stirred, cold (0° C.) solution of methyl 2-amino-5-ethyl-1,3-thiazole-4-carboxylate (186 mg, 1.0 mmol), CuSO₄.5H₂O (330 mg, 1.32 mmol), NaCl (260 mg, 4.45 mmol) and H₂SO₄ (5.5 mL) in water (7.5 mL). The mixture was stirred at 0° C. for 45 min and allowed to warm up to room temperature where it stirred further for 1 h before CuCl (118 mg) was added. This mixture was stirred further at room temperature for 16 h before it was diluted with brine and extracted with ether twice. The organic layers were combined, dried over MgSO₄ and concentrated to give methyl 2-chloro-5-ethylthiazole-4-carboxylate (i.e. Cap-172, step a) (175 mg, 85%) as an orange oil (80% pure) which was used directly in the next reaction. R_(t)=1.99 min (Cond.—MD1); LC/MS: Anal. Calcd. for [M+H]⁺ C₇H₉ClNO₂S: 206.01; found: 206.05.

Cap-172

To a solution of methyl 2-chloro-5-ethylthiazole-4-carboxylate (175 mg) in THF/H₂O/MeOH (20 mL/3 mL/12 mL) was added LiOH (305 mg, 12.76 mmol). The mixture was stirred at room temperature overnight before it was concentrated down and neutralized with 1N HCl in ether (25 mL). The residue was extracted twice with ethyl acetate and the organic layers were combined, dried over MgSO₄ and evaporated to yield Cap-172 (60 mg, 74%) as a red solid which was used without further purification. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 13.03-13.42 (1 H, m), 3.16 (2 H, q, J=7.4 Hz), 1.23 (3 H, t, J=7.5 Hz). R_(t)=1.78 min (Cond.—MD1); LC/MS: Anal. Calcd. for [M+H]⁺ C₆H₇ClNO₂S: 191.99; found: 191.99.

The following diazotization step was adapted from Barton, A.; Breukelman, S. P.; Kaye, P. T.; Meakins, G. D.; Morgan, D. J. J. C. S. Perkin Trans I 1982, 159-164: A solution of NaNO₂ (150 mg, 2.17 mmol) in water (1.0 mL) was added dropwise to a stirred, cold (0° C.) solution of methyl 2-amino-5-ethyl-1,3-thiazole-4-carboxylate (186 mg, 1.0 mmol) in 50% H₃PO₂ (3.2 mL). The mixture was stirred at 0° C. for 1 h and allowed to warm up to room temperature where it stirred further for 2 h. After recooling to 0° C., the mixture was treated slowly with a solution of NaOH (85 mg) in water (10 mL). The mixture was then diluted with saturated NaHCO₃ solution and extracted twice with ether. The organic layers were combined, dried over MgSO₄ and concentrated to give methyl 5-ethylthiazole-4-carboxylate (i.e. Cap-173, step a) (134 mg, 78%) as an orange oil (85% pure) which was used directly in the next reaction. R_(t)=1.58 min (Cond.—MD1); LC/MS: Anal. Calcd. for [M+H]⁺ C₇H₁₀NO₂S: 172.05; found: 172.05.

Cap-473

To a solution of methyl 5-ethylthiazole-4-carboxylate (134 mg) in THF/H₂O/MeOH (18 mL/2.7 mL/11 mL) was added LiOH (281 mg, 11.74 mmol). The mixture was stirred at room temperature overnight before it was concentrated down and neutralized with 1N HCl in ether (25 mL). The residue was extracted twice with ethyl acetate and the organic layers were combined, dried over MgSO₄ and evaporated to yield Cap-173 (90 mg, 73%) as an orange solid which was used without further purification. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 12.74-13.04 (1 H, m), 3.20 (2 H, q, J=7.3 Hz), 1.25 (3 H, t, J=7.5 Hz). R_(t)=1.27 min (Cond.—MD1); LC/MS: Anal. Calcd. for [M+H]⁺ C₆H₈NO₂S: 158.03; found: 158.04.

Triflic anhydride (5.0 g, 18.0 mmol) was added dropwise to a cold (0° C.) solution of methyl 3-hydroxypicolinate (2.5 g, 16.3 mmol) and TEA (2.5 mL, 18.0 mmol) in CH₂Cl₂ (80 mL). The mixture was stirred at 0° C. for 1 h before it was allowed to warm up to room temperature where it stirred for an additional 1 h. The mixture was then quenched with saturated NaHCO₃ solution (40 mL) and the organic layer was separated, washed with brine, dried over MgSO₄ and concentrated to give methyl 3-(trifluoromethylsulfonyloxy)picolinate (i.e. Cap-174, step a) (3.38 g, 73%) as a dark brown oil (>95% pure) which was used directly without further purification. ¹H NMR (300 MHz, CDCl₃) δ ppm 8.72-8.79 (1 H, m), 7.71 (1 H, d, J=1.5 Hz), 7.58-7.65 (1 H, m), 4.04 (3 H, s). R_(t)=1.93 min (Cond.—MD1); LC/MS: Anal. Calcd. for [M+H]⁺ C₈H₇F₃NO₅S: 286.00; found: 286.08.

Cap-174

To a solution of methyl 3-(trifluoromethylsulfonyloxy)picolinate (570 mg, 2.0 mmol) in DMF (20 mL) was added LiCl (254 mg, 6.0 mmol), tributyl(vinyl)stannane (761 mg, 2.4 mmol) and bis(triphenylphosphine)palladium dichloride (42 mg, 0.06 mmol). The mixture was heated at 100° C. overnight before a saturated solution of KF (20 mL) was added to the reaction mixture at room temperature. This mixture was stirred for 4 h before it was filtered through diatomaceous earth (Celite®) and the pad was washed with ethyl acetate. The aqueous phase of the filtrate was then separated and concentrated down in vacuo. The residue was treated with 4N HCl in dioxanes (5 mL) and the resulting mixture was extracted with methanol, filtered and evaporated to afford Cap-174 (260 mg) as a green solid which was slightly contaminated with inorganic salts but was used without further purification. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 8.21 (1 H, d, J=3.7 Hz), 7.81-7.90 (1 H, m), 7.09 (1 H, dd, J=7.7, 4.8 Hz), 6.98 (1 H, dd, J=17.9, 11.3 Hz), 5.74 (1 H, dd, J=17.9, 1.5 Hz), 5.20 (1 H, d, J=11.0 Hz). R_(t)=0.39 min (Cond.—MD1); LC/MS: Anal. Calcd. for [M+H]⁺ C₈H₈NO₂: 150.06; found: 150.07.

To a solution of methyl 3-(trifluoromethylsulfonyloxy)picolinate (i.e. Cap 173, step a) (570 mg, 2.0 mmol), an intermediate in the preparation of Cap-174, in DMF (20 mL) was added LiCl (254 mg, 6.0 mmol), tributyl(vinyl)stannane (761 mg, 2.4 mmol) and bis(triphenylphosphine)palladium dichloride (42 mg, 0.06 mmol). The mixture was heated at 100° C. for 4 h before the solvent was removed in vacuo. The residue was taken up in acetonitrile (50 mL) and hexanes (50 mL) and the resulting mixture was washed twice with hexanes. The acetonitrile layer was then separated, filtered through Celite, and evaporated. Purification of the residue by flash chromatography on a Horizon instrument (gradient elution with 25% ethyl acetate in hexanes to 65% ethyl acetate in hexanes) afforded methyl 3-vinylpicolinate (i.e. Cap-175, step a) (130 mg, 40%) as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ ppm 8.60 (1 H, dd, J=4.6, 1.7 Hz), 7.94 (1 H, d, J=7.7 Hz), 7.33-7.51 (2 H, m), 5.72 (1 H, d, J=17.2 Hz), 5.47 (1 H, d, J=11.0 Hz), 3.99 (3 H, s). R_(t)=1.29 min (Cond.—MD1); LC/MS: Anal. Calcd. for [M+H]⁺ C₉H₁₀NO₂: 164.07; found: 164.06.

Palladium on carbon (10%, 25 mg) was added to a solution of methyl 3-vinylpicolinate (120 mg, 0.74 mmol) in ethanol (10 mL). The suspension was stirred at room temperature under an atmosphere of hydrogen for 1 h before it was filtered through Celite and the pad of diatomaceous earth (Celite®) was washed with methanol. The filtrate was concentrated down to dryness to yield methyl 3-ethylpicolinate (i.e. Cap-175, step b) which was taken directly into the next reaction. R_(t)=1.15 min (Cond.—MD1); LC/MS: Anal. Calcd. for [M+H]⁺ C₉H₁₂NO₂: 166.09; found: 166.09.

Cap-175

To a solution of methyl 3-ethylpicolinate in THF/H₂O/MeOH (5 mL/0.75 mL/3 mL) was added LiOH (35 mg, 1.47 mmol). The mixture was stirred at room temperature for 2 d before additional LiOH (80 mg) was added. After an additional 24 h at room temperature, the mixture was filtered and the solvent was removed in vacuo. The residue was then treated with 4N HCl in dioxanes (5 mL) and the resulting suspension was concentrated down to dryness to yield Cap-175 as a yellow solid which was used without further purification. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 8.47 (1 H, dd, J=4.8, 1.5 Hz), 7.82-7.89 (1 H, m), 7.53 (1 H, dd, J=7.7, 4.8 Hz), 2.82 (2 H, q, J=7.3 Hz), 1.17 (3 H, t, J=7.5 Hz). R_(t)=0.36 min (Cond.—MD1); LC/MS: Anal. Calcd. for [M+H]⁺ C₈H₁₀NO₂: 152.07; found: 152.10.

A solution of 1,4-dioxaspiro[4.5]decan-8-one (15 g, 96 mmol) in EtOAc (150 mL) was added to a solution of methyl 2-(benzyloxycarbonylamino)-2-(dimethoxyphosphoryl)acetate (21.21 g, 64.0 mmol) in 1,1,3,3-tetramethylguanidine (10.45 mL, 83 mmol) and EtOAc (150 mL). The resulting solution was the stirred at ambient temperature for 72 h and then it was diluted with EtOAc (25 mL). The organic layer was washed with 1N HCl (75 mL), H₂O (100 mL) and brine (100 mL), dried (MgSO₄), filtered and concentrated. The residue was purified via Biotage (5% to 25% EtOAc/Hexanes; 300 g column). The combined fractions containing the product were then concentrated under vacuum and the residue was re-crystallized from hexanes/EtOAc to give white crystals that corresponded to methyl 2-(benzyloxycarbonylamino)-2-(1,4-dioxaspiro[4.5]decan-8-ylidene)acetate (6.2 g) ¹H NMR (400 MHz, CDCl₃-d) δ ppm 7.30-7.44 (5 H, m), 6.02 (1 H, br. s.), 5.15 (2 H, s), 3.97 (4 H, s), 3.76 (3 H, br. s.), 2.84-2.92 (2 H, m), 2.47 (2 H, t, J=6.40 Hz), 1.74-1.83 (4 H, m). LC (Cond. OL1): R_(t)=2.89 min. LC/MS: Anal. Calcd. For [M+N]⁺ C₁₉H₂₃NNaO₆: 745.21; found: 745.47.

Ester Cap 176, step b was prepared from alkene Cap 176, step a according to the method of Burk, M. J.; Gross, M. F. and Martinez J. P. (J. Am. Chem. Soc., 1995, 127, 9375-9376 and references therein): A 500 mL high-pressure bottle was charged with alkene Cap 176, step a (3.5 g, 9.68 mmol) in degassed MeOH (200 mL) under a blanket of N₂. The solution was then charged with (−)-1,2-Bis((2S,5S)-2,5-dimethylphospholano)ethane cyclooctadiene)rhodium (I) tetrafluoroborate (0.108 g, 0.194 mmol) and the resulting mixture was flushed with N₂ (3×) and charged with H₂ (3×). The solution was shaken vigorously under 70 psi of H₂ at ambient temperature for 72 h. The solvent was removed under reduced pressure and the remaining residue was taken up in EtOAc. The brownish solution was then filtered through a plug of Silica Gel and eluted with EtOAc. The solvent was concentrated under vacuum to afford a clear oil corresponding to ester Cap 176, step b (3.4 g). ¹H NMR (500 MHz, CDCl₃-d) δ ppm 7.28-7.43 (5 H, m), 5.32 (1 H, d, J=9.16 Hz), 5.06-5.16 (2 H, m), 4.37 (1 H, dd, J=9.00, 5.04 Hz), 3.92 (4 H, t, J=3.05 Hz), 3.75 (3 H, s), 1.64-1.92 (4 H, m), 1.37-1.60 (5 H, m). LC (Cond. OL1): R_(t)=1.95 min. LC/MS: Anal. Calcd. For [M+H]⁺ C₁₉H₂₆NO₆: 364.18; found: 364.27.

Ester Cap 176, step b (4.78 g, 13.15 mmol) was dissolved in THF (15 mL) followed by sequential addition of water (10 mL), glacial acetic acid (26.4 mL, 460 mmol) and dichloroacetic acid (5.44 mL, 65.8 mmol). The resulting mixture was stirred for 72 h at ambient temperature, and the reaction was quenched by slow addition of solid Na₂CO₃ with vigorous stirring until the release of gas was no longer visible. Crude product was extracted into 10% ethyl acetate-dichloromethane and the organic layers were combined, dried (MgSO₄) filtered and concentrated. The resulting residue was purified via Biotage (0 to 30% EtOAc/Hex; 25 g column) to afford ketone Cap 176, step c (3.86 g) as a clear oil. ¹H NMR (400 MHz, CDCl₃-d) δ ppm 7.28-7.41 (5 H, m), 5.55 (1 H, d, Hz), 5.09 (2 H, s), 4.46 (1 H, dd, J=8.16, 5.14 Hz), 3.74 (3 H, s), 2.18-2.46 (5 H, m), 1.96-2.06 (1 H, m), 1.90 (1 H, ddd, J=12.99, 5.96, 2.89 Hz), 1.44-1.68 (2 H, m, J=12.36, 12.36, 12.36, 12.36, 4.77 Hz). LC (Cond. OL1): R_(t)=1.66 min. LC/MS: Anal. Calcd. For [M+Na]⁺ C₁₇H₂₁NNaO_(s): 342.13; found: 342.10.

Deoxo-Fluor® (3.13 mL, 16.97 mmol) was added to a solution of ketone Cap 176, step c (2.71 g, 8.49 mmol) in CH₂Cl₂ (50 mL) followed by addition of a catalytic amount of EtOH (0.149 mL, 2.55 mmol). The resulting yellowish solution was stirred at rt overnight. The reaction was quenched by addition of sat. aq. NaHCO₃ (25 mL) and the mixture was extracted with EtOAc (3×75 mL)). The combined organic layers were dried (MgSO₄), filtered and dried to give a yellowish oil. The residue was purified via Biotage chromatography (2% to 15% EtOAc/Hex; 90 g column) and a white solid corresponding to the difluoro amino acid dilforide Cap 176, step d (1.5 g) was recovered. ¹H NMR (400 MHz, CDCl₃-d) δ ppm 7.29-7.46 (5 H, m), 5.34 (1 H, d, J=8.28 Hz), 5.12 (2 H, s), 4.41 (1H, dd, J=8.66, 4.89 Hz), 3.77 (3 H, s), 2.06-2.20 (2 H, m), 1.83-1.98 (1 H, m), 1.60-1.81 (4 H, m), 1.38-1.55 (2 H, m). ¹⁹F NMR (376 MHz, CDCl₃-d) δ ppm −92.15 (1 F, d, J=237.55 Hz), −102.44 (1F, d, J=235.82 Hz). LC (Cond. OL1): R_(t)=1.66 min. LC/MS: Anal. Calcd. For [2M+Na]⁺ C₃₄H₄₂F₄N₂NaO₈: 705.28; found: 705.18.

Difluoride Cap 176, step d (4 g, 11.72 mmol) was dissolved in MeOH (120 mL) and charged with Pd/C (1.247 g, 1.172 mmol). The suspension was flushed with N₂ (3×) and the reaction mixture was placed under 1 atm of H₂ (balloon). The mixture was stirred at ambient temperature for 48 h. The suspension was then filtered though a plug of Celite and concentrated under vacuum to give an oil that corresponded to amino acid Cap 176, step e (2.04 g) and that was used without further purification. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.62 (3 H, s), 3.20 (1 H, d, J=5.77 Hz), 1.91-2.09 (2 H, m), 1.50-1.88 (7 H, m), 1.20-1.45 (2 H, m). ¹⁹F NMR (376 MHz, DMSO-d₆) ● ppm −89.39 (1 F, d, J=232.35 Hz), −100.07 (1 F, d, J=232.35 Hz). ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 175.51 (1 C, s), 124.10 (1 C, t, J=241.21, 238.90 Hz), 57.74 (1 C, s), 51.39 (1 C, s), 39.23 (1 C, br. s.), 32.02-33.83 (2 C, m), 25.36 (1 C, d, J=10.02 Hz), 23.74 (1 C, d, J=9.25 Hz). LC (Cond. OL2): R_(t)=0.95 min. LC/MS: Anal. Calcd. For [2M+H]⁺ C₁₈H₃₁F₄N₂O₂: 415.22; found: 415.40.

Methyl chloroformate (1.495 mL, 19.30 mmol) was added to a solution of amino acid Cap 176, step e (2 g, 9.65 mmol) and DIEA (6.74 mL, 38.6 mmol) in CH₂Cl₂ (100 mL). The resulting solution was stirred at rt for 3 h and volatiles were removed under reduced pressure. The residue was purified via Biotage (0% to 20% EtOAc/Hex; 90 g column). A clear oil that solidified upon standing under vacuum and corresponding to carbamate Cap-176, step f (2.22 g) was recovered. ¹H NMR (500 MHz, CDCl₃-d) δ ppm 5.27 (1 H, d, J=8.55 Hz), 4.39 (1 H, dd, J=8.85, 4.88 Hz), 3.77 (3 H, s), 3.70 (3 H, s), 2.07-2.20 (2 H, m), 1.84-1.96 (1 H, m), 1.64-1.82 (4 H, m), 1.39-1.51 (2 H, m). ¹⁹F NMR (471 MHz, CDCl₃-d) δ ppm −92.55 (1 F, d, J=237.13 Hz), −102.93 (1 F, d, J=237.12 Hz). ¹³C NMR (126 MHz, CDCl₃-d) δ ppm 171.97 (1 C, s), 156.69 (1 C, s), 119.77-125.59 (1 C, m), 57.24 (1 C, br. s.), 52.48 (1 C, br. s.), 52.43 (1 C, s), 39.15 (1 C, s), 32.50-33.48 (2 C, m), 25.30 (1 C, d, J=9.60 Hz), 24.03 (1 C, d, J=9.60 Hz). LC (Cond. OL1): R_(t)=1.49 min. LC/MS: Anal. Calcd. For [M+Na]⁺ C₁₁H₁₇F₂NNaO₄: 288.10; found: 288.03.

Cap-176

A solution of LiOH (0.379 g, 15.83 mmol) in Water (25 mL) was added to a solution of carbamate Cap-176, step f (2.1 g, 7.92 mmol) in THF (75 mL) and the resulting mixture was stirred at ambient temperature for 4 h. THF was removed under vacuum and the remaining aqueous phase was acidified with 1N HCl solution (2 mL) and then extracted with EtOAc (2×50 mL). The combined organic layers were dried (MgSO₄), filtered and concentrated to give a white foam corresponding to Cap-176 (1.92 g). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.73 (1 H, s), 7.50 (1 H, d, J=8.78 Hz), 3.97 (1 H, dd, J=8.53, 6.02 Hz), 3.54 (3 H, s), 1.92-2.08 (2 H, m), 1.57-1.90 (5 H, m), 1.34-1.48 (1 H, m), 1.27 (1 H, qd, J=12.72, 3.26 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm −89.62 (1 F, d, J=232.35 Hz), −99.93 (1 F, d, J=232.35 Hz). LC (Cond. OL2): R_(t)=0.76 min. LC/MS: Anal. Calcd. For [M−H]⁺ C₁₀H₁₄F₂NO₄: 250.09; found: 250.10.

Biological Activity

An HCV Replion assay was utilized in the present disclosure, and was prepared, conducted and validated as described in commonly owned PCT/US2006/022197 and in O'Boyle et. al. Antimicrob Agents Chemother. 2005 April; 49(4):1346-53. Assay methods incorporating luciferase reporters have also been used as described (Apath.com).

HCV-neo replicon cells and replicon cells containing mutations in the NS5A region were used to test the currently described family of compounds. The compounds were determined to have more than 10-fold less inhibitory activity on cells containing mutations than wild-type cells. Thus, the compounds of the present disclosure can be effective in inhibiting the function of the HCV NS5A protein and are understood to be as effective in combinations as previously described in application PCT/US2006/022197 and commonly owned WO/04014852. Further, the compounds of the present disclosure can be effective against the HCV 1b genotype. It should also be understood that the compounds of the present disclosure can inhibit multiple genotypes of HCV. Table 2 shows the EC₅₀ (Effective 50% inhibitory concentration) values of representative compounds of the present disclosure against the HCV 1b genotype. In one embodiment, compounds of the present disclosure are inhibitory versus 1a, 1b, 2a, 2b, 3a, 4a, and 5a genotypes.

TABLE 2 1b EC₅₀ Example in μM Name 1 0.000034 Methyl ((1S)-1-(((2S)-2-(4-(6-(2-((2S)-1- ((2S)-2-((methoxycarbonyl)amino)-3- methylbutanoyl)-2-pyrrolidinyl)-1H- imidazol-4-yl) [1,3]thiazolo[5,4- f] [1,3]benzothiazol-2-yl)-1H-imidazol- 2-yl)-1-pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate 2 0.000025 Dimethyl ([1,3]thiazolo[5,4- f] [1,3]benzothiazole-2,6-diylbis(1H- imidazole-4,2-diyl(2S)-2,1- pyrrolidinediyl((1R)-2-oxo-1-phenyl- 2,1-ethanediyl)))biscarbamate 3 0.35 Methyl ((1R)-1-(((2S)-2-(4-(6-(2-((2S)- 1-((2R)-2-((methoxycarbonyl)amino)-3- methylbutanoyl)-2-pyrrolidinyl)-1H- imidazol-4-yl) [1,3]thiazolo[5,4- f] [1,3]benzothiazol-2-yl)-1H-imidazol- 2-yl)-1-pyrrolidinyl)carbonyl)-2- methylpropyl)carbamate

The compounds of the present disclosure may inhibit HCV by mechanisms in addition to or other than NS5A inhibition. In one embodiment the compounds of the present disclosure inhibit HCV replicon and in another embodiment the compounds of the present disclosure inhibit NS5A.

It will be evident to one skilled in the art that the present disclosure is not limited to the foregoing illustrative examples, and that it can be embodied in other specific forms without departing from the essential attributes thereof. It is therefore desired that the examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing examples, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. 

1. A compound of Formula (I) or Formula (II):

or a pharmaceutically acceptable salt thereof, wherein: Z is sulfur (S); L¹ is

L² is

R¹ is hydrogen or —C(O)R^(x); R² is hydrogen or —C(O)R^(y); R^(x) and R^(y) are each independently selected from cycloalkyl, heteroaryl, heterocyclyl, alkoxy, and alkyl, said alkyl being substituted by one or more substituents independently selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(c)R^(d), wherein any said aryl and heteroaryl may optionally be substituted with one or more substituents independently selected from alkyl, haloalkyl, arylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, OR⁵, —NR^(a)R^(b), (NR^(a)R^(b))alkyl, and (MeO)(HO)P(O)O—, and wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴; R³ is hydrogen, alkyl, or arylalkyl; R⁴ is alkyl or arylalkyl; R⁵ is hydrogen, alkyl, or arylalkyl; R^(a) and R^(b) are independently selected from hydrogen, alkyl, cycloalkyl, arylalkyl, heteroaryl, —C(O)R⁶, —C(O)OR⁷, —C(O)NR^(c)R^(d), and (NR^(c)R^(d))alkyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring or bridged bicyclic ring structure, wherein said five- or six-membered ring or bridged bicyclic ring structure optionally may contain one or two additional heteroatoms independently selected from nitrogen, oxygen, and sulfur and may contain one, two, or three substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, aryl, hydroxyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, and halogen; R⁶ is alkyl; R⁷ is alkyl, arylalkyl, cycloalkyl, or haloalkyl; and R^(c) and R^(d) are independently selected from hydrogen, alkyl, arylalkyl, and cycloalkyl.
 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L¹ is

and L² is


3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L¹ is

and L² is


4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L¹ is

and L² is


5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L¹ is

and L² is


6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R¹ is —C(O)R^(x); R² is —C(O)R^(y); and R^(x) and R^(y) are independently alkyl substituted by at least one —NR^(a)R^(b), characterized by Formula (A):

wherein: m is 0 or 1; R⁸ is hydrogen or alkyl; R⁹ is selected from hydrogen, cycloalkyl, aryl, heteroaryl, heterocyclyl, and alkyl optionally substituted with a substituent selected from aryl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, heterobicyclyl, —OR³, —C(O)OR⁴, —NR^(a)R^(b), and —C(O)NR^(c)R^(d), wherein any said aryl and heteroaryl may optionally be substituted with one or more substituents independently selected from alkyl, haloalkyl, arylakyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, —C(O)OR⁴, OR⁵, —NR^(a)R^(b), (NR^(a)R^(b))alkyl, and (MeO)(HO)P(O)O—, and wherein any said cycloalkyl and heterocyclyl may optionally be fused onto an aromatic ring and may optionally be substituted with one or more substituents independently selected from alkyl, hydroxyl, halogen, aryl, —NR^(a)R^(b), oxo, and —C(O)OR⁴; and R³, R⁴, R⁵, R^(a), R^(b), R^(c), and R^(d) are defined as in claim
 1. 7. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein: m is 0; R⁸ is hydrogen or C₁ to C₄ alkyl; R⁹ is selected from hydrogen, C₁ to C₆ alkyl optionally substituted with —OR¹², C₃ to C₆ cycloalkyl, allyl, —CH₂C(O)NR^(c)R^(d), (NR^(c)R^(d)) alkyl,

wherein j is 0 or 1; k is 1, 2, or 3; n is 0 or an integer selected from 1 through 4; each R¹⁰ is independently hydrogen, C₁ to C₄ alkyl, C₁ to C₄ haloalkyl, halogen, nitro, —OBn, or (MeO) (OH)P(O)O—; R¹¹ is hydrogen, C₁ to C₄ alkyl, or benzyl; R¹² is hydrogen, C₁ to C₄ alkyl, or benzyl; R^(a) is hydrogen or C₁ to C₄ alkyl; R^(b) is C₁ to C₄ alkyl, C₃ to C₆ cycloalkyl, benzyl, 3-pyridyl, pyrimidin-5-yl, acetyl, —C(O)OR⁷, or —C(O)NR^(c)R^(d); R⁷ is C₁ to C₄ alkyl or C₁ to C₄ haloalkyl; R^(c) is hydrogen or C₁ to C₄ alkyl; and R^(d) is hydrogen, C₁ to C₄ alkyl, or C₃ to C₆ cycloalkyl.
 8. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein: m is 0; R⁸ is hydrogen; R⁹ is phenyl optionally substituted with one up to five substituents independently selected from C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, halogen, C₁ to C₆ alkoxy, hydroxyl, cyano, and nitro; and NR^(a)R^(b) is a heterocyclyl or heterobicyclyl group selected from:

wherein n is 0, 1, or 2; each R¹³ is independently selected from C₁ to C₆ alkyl, phenyl, trifluoromethyl, halogen, hydroxyl, methoxy, and oxo; and R¹⁴ is C₁ to C₆ alkyl, phenyl, benzyl, or —C(O)OR¹⁵ group, wherein R¹⁵ is C₁ to C₄ alkyl, phenyl, or benzyl.
 9. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein: m is 1; R⁸ is hydrogen; R⁹ is C₁ to C₆ alkyl, arylalkyl, or heteroarylalkyl; R^(a) is hydrogen; and R^(b) is —C(O)OR⁷, wherein R⁷ is C₁ to C₆ alkyl.
 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R¹ is —C(O)R^(x); R² is —C(O)R^(y); and R^(x) and R^(y) are heteroaryl or heterocyclyl independently selected from:

wherein n is 0 or an integer selected from 1 through 4; each R¹³ is independently selected from hydrogen, C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, phenyl, benzyl, C₁ to C₆ alkoxy, C₁ to C₄ haloalkoxy, heterocyclyl, halogen, NR^(c)R^(d), hydroxyl, cyano, and oxo, where R^(c) and R^(d) are independently hydrogen or C₁ to C₄ alkyl; and R¹⁴ is hydrogen (H), C₁ to C₆ alkyl, benzyl, or —C(O)OR⁴, wherein R⁴ is C₁ to C₆ alkyl.
 11. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R¹ is —C(O)R^(x); R² is —C(O)R^(y); and R^(x) and R^(y) are cycloalkyl independently selected from:

wherein j is 0, 1, 2, or 3; k is 0, 1, or 2; n is 0 or an integer selected from 1 though 4; each R¹³ is independently selected from hydrogen, C₁ to C₆ alkyl, C₁ to C₄ haloalkyl, C₁ to C₆ alkoxy, halogen, hydroxyl, cyano, and nitro; and R^(a) and R^(b) are independently hydrogen, C₁ to C₆ alkyl, or —C(O)OR⁷, wherein R⁷ is C₁ to C₆ alkyl.
 12. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R¹ is —C(O)R^(x); R² is —C(O)R^(y); and R^(x) and R^(y) are independently arylalkyl optionally substituted with (NR^(a)R^(b))alkyl, wherein R^(a) and R^(b) are independently hydrogen, C₁ to C₆ alkyl, or benzyl, or alternatively, R^(a) and R^(b), together with the nitrogen atom to which they are attached, form a five- or six-membered ring selected from

 and

 wherein R¹⁵ is hydrogen, C₁ to C₆ alkyl, or benzyl.
 13. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R¹ and R² are the same and are selected from the group consisting of:

wherein a squiggle bond (

) in the structure indicates that a stereogenic center to which the bond is attached can take either (R)- or (S)-configuration so long as chemical bonding principles are not violated.
 14. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R¹ is —C(O)R^(x); R² is —C(O)R^(y); and R^(x) and R^(y) are both t-butoxy.
 15. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R¹ and R² are both hydrogen.
 16. A compound, or a pharmaceutically acceptable salt thereof, selected from the group consisting of: di-tert-butyl (2S,2′S)-2,2′-(benzo[1,2-d:4,5-d′]bis[1,3]thiazole-2,6-diylbis(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-5,2-diyl))di(1-pyrrolidinecarboxylate); 2,6-bis(2-((2S)-2-pyrrolidinyl)-1H-imidazol-5-yl)benzo[1,2-d:4,5-d′]bis[1,3]thiazole; methyl ((1S)-1-(((2S)-2-(4-(6-(2-((2S)-1-((2S)-2-((methoxycarbonyl)amino)-3-methylbutanoyl)-2-pyrrolidinyl)-1H-imidazol-4-yl)[1,3]thiazolo[5,4-f][1,3]benzothiazol-2-yl)-1H-imidazol-2-yl)-1-pyrrolidinyl)carbonyl)-2-methylpropyl)carbamate; dimethyl ([1,3]thiazolo[5,4-f][1,3]benzothiazole-2,6-diylbis(1H-imidazole-4,2-diyl(2S)-2,1-pyrrolidinediyl((1R)-2-oxo-1-phenyl-2,1-ethanediyl)))biscarbamate; methyl ((1R)-1-(((2S)-2-(4-(6-(2-((2S)-1-((2R)-2-((methoxycarbonyl)amino)-3-methylbutanoyl)-2-pyrrolidinyl)-1H-imidazol-4-yl)[1,3]thiazolo[5,4-f][1,3]-benzothiazol-2-yl)-1H-imidazol-2-yl)-1-pyrrolidinyl)carbonyl)-2-methylpropyl)carbamate; and corresponding stereoisomers and tautomers thereof.
 17. A composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 18. A method of treating an HCV infection in a patient, comprising administering to the patient a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof. 